Modulation of apolipoprotein ciii (apociii) expression

ABSTRACT

Provided herein are methods, compounds, and compositions for reducing expression of ApoCIII mRNA and protein in an animal. Also provided herein are methods, compounds, and compositions for increasing HDL levels and/or improving the ratio of TG to HDL and reducing plasma lipids and plasma glucose in an animal. Such methods, compounds, and compositions are useful to treat, prevent, delay, or ameliorate any one or more of cardiovascular disease or metabolic disorder, or a symptom thereof.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0130USC1SEQ_ST25.TXT, created on Aug. 7, 2015 which is 8 Kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Provided herein are methods, compounds, and compositions for reducingexpression of Apolipoprotein CIII (ApoCIII) mRNA and protein, andincreasing HDL or HDL activity in an animal. Also, provided herein aremethods, compounds, and compositions for an ApoCIII inhibitor forreducing ApoCIII related diseases or conditions in an animal.

BACKGROUND

Lipoproteins are globular, micelle-like particles that consist of anon-polar core of acylglycerols and cholesteryl esters surrounded by anamphiphilic coating of protein, phospholipid and cholesterol.Lipoproteins have been classified into five broad categories on thebasis of their functional and physical properties: chylomicrons, verylow density lipoproteins (VLDL), intermediate density lipoproteins(IDL), low density lipoproteins (LDL), and high density lipoproteins(HDL). Chylomicrons transport dietary lipids from intestine to tissues.VLDLs, IDLs and LDLs all transport triacylglycerols and cholesterol fromthe liver to tissues. HDLs transport endogenous cholesterol from tissuesto the liver.

Lipoprotein particles undergo continuous metabolic processing and havevariable properties and compositions. Lipoprotein densities increasewithout increasing particle diameter because the density of their outercoatings is less than that of the inner core. The protein components oflipoproteins are known as apolipoproteins. At least nine apolipoproteinsare distributed in significant amounts among the various humanlipoproteins.

Apolipoprotein C-III (ApoCIII) is a constituent of HDL and oftriglyceride (TG)-rich lipoproteins. Elevated ApoCIII is associated withhypertriglyceridemia. Accordingly, ApoCIII has a role inhypertriglyceridemia, a risk factor for coronary artery disease(Davidsson et al., J. Lipid Res. 2005. 46: 1999-2006). ApoCIII slowsclearance of triglyceride-rich lipoproteins by inhibiting lipolysis,both through inhibition of lipoprotein lipase and by interfering withlipoprotein binding to cell-surface glycosaminoglycan matrix (Shachter,Curr. Opin. Lipidol., 2001, 12, 297-304).

The gene encoding human apolipoprotein C-III (also called APOC3,APOC-III, ApoCIII, and APO C-III) was cloned in 1984 by three researchgroups (Levy-Wilson et al., DNA, 1984, 3, 359-364; Protter et al., DNA,1984, 3, 449-456; Sharpe et al., Nucleic Acids Res., 1984, 12,3917-3932). The coding sequence is interrupted by three introns (Protteret al., DNA, 1984, 3, 449-456). The human ApoCIII gene is locatedapproximately 2.6 kb to the 3′ direction of the apolipoprotein A-1 geneand these two genes are convergently transcribed (Karathanasis, Proc.Natl. Acad. Sci. U.S.A, 1985, 82, 6374-6378). Also cloned was a variantof human apolipoprotein C-III with a Thr74 to Ala74 mutation from apatient with unusually high level of serum apolipoprotein C-III. As theThr74 is O-glycosylated, the Ala74 mutant therefore resulted inincreased levels of serum ApoCIII lacking the carbohydrate moiety (Maedaet al., J. Lipid Res., 1987, 28, 1405-1409). Other variants orpolymorphisms that modulated Apo CIII expression were later identified.Some of the polymorphsims elevated ApoCIII. Elevated ApoCIII levels wereassociated with elevated triglyceride (TG) levels and diseases such ascardiovascular disease, metabolic syndrome, obesity and diabetes (Chanet al., Int J Clin Pract, 2008, 62:799-809; Onat et al.,Atherosclerosis, 2003, 168:81-89; Mendivil et al., Circulation, 2011,124:2065-2072).

Five polymorphisms have been identified in the promoter region of thegene: C (at position −641 of the gene) to A, G (at position −630 of thegene) to A, T (at position −625 of the gene) to deletion, C (at position−482 of the gene) to T and T (at position −455 of the gene) to C. All ofthese polymorphisms are in linkage disequilibrium with the SstIpolymorphism in the 3′ untranslated region. The SstI polymorphic sitedistinguishes the S1 and S2 alleles and the S2 allele has beenassociated with elevated plasma triglyceride levels (Dammerman et al.,Proc. Natl. Acad. Sci. U.S.A, 1993, 90, 4562-4566). The ApoCIII promoteris downregulated by insulin and this polymorphic site abolishes theinsulin regulation. Thus the potential overexpression of ApoCIIIresulting from the loss of insulin regulation may be a contributingfactor to the development of hypertriglyceridemia associated with the S2allele (Li et al., J. Clin. Invest., 1995, 96, 2601-2605). The T (atposition −455 of the gene) to C polymorphism has been associated with anincreased risk of coronary artery disease (Olivieri et al., J. LipidRes., 2002, 43, 1450-1457). Other polymorphisms in the human ApoCIIIgene that have been associated with elevated ApoCIII and/or triglycerideexpression include: C (at position 1100) to T, C (at position 3175) toG, T (at position 3206) to G, C (at positions 3238) to G, etc. (Tilly etal., J. Lipid Res., 2003, 44:430-436; Waterworth et al., ArteriosclerThromb Vasc Biol, 2000, 20:2663-2669; Petersen et al., N Engl J Med,2010, 362:1082-1089).

In addition to insulin, other regulators of ApoCIII gene expression havebeen identified. A response element for the nuclear orphan receptorrev-erb alpha has been located at positions −23 to −18 of the gene, inthe ApoCIII promoter region. Rev-erb alpha decreases ApoCIII promoteractivity (Raspe et al., J. Lipid Res., 2002, 43, 2172-2179). The ApoCIIIpromoter region from positions −86 to −74 of the gene is recognized bytwo nuclear factors CIIIb1 and CIIIB2 (Ogami et al., J. Biol. Chem.,1991, 266, 9640-9646). ApoCIII expression is also upregulated byretinoids acting via the retinoid X receptor, and alterations inretinoid X receptor abundance affects ApoCIII transcription (Vu-Dac etal., J. Clin. Invest., 1998, 102, 625-632). Specificity protein 1 (Sp1)and hepatocyte nuclear factor-4 (HNF-4) have been shown to worksynergistically to transactivate the apolipoprotein C-III promoter viathe HNF-4 binding site (Kardassis et al., Biochemistry, 2002, 41,1217-1228). HNF-4 also works in conjunction with SMAD3-SMAD4 totransactivate the ApoCIII promoter (Kardassis et al., J. Biol. Chem.,2000, 275, 41405-41414).

Transgenic and knockout mice have further defined the role of ApoCIII inlipolysis. Overexpression of ApoCIII in transgenic mice leads tohypertriglyceridemia and impaired clearance of VLDL-triglycerides (deSilva et al., J. Biol. Chem., 1994, 269, 2324-2335; Ito et al., Science,1990, 249, 790-793). Knockout mice with a total absence of the ApoCIIIprotein exhibited significantly reduced plasma cholesterol andtriglyceride levels compared with wild-type mice and were protected frompostprandial hypertriglyceridemia (Maeda et al., J. Biol. Chem., 1994,269, 23610-23616).

Total plasma ApoCIII levels have been identified as a major determinantof serum triglycerides, and epidemiological studies have demonstratedthat ApoCIII and ApoB lipoproteins that have ApoCIII as a componentindependently predict coronary heart disease (Sacks et al., Circulation.2000. 102: 1886-1892; Lee et al., Arterioscler Thromb Vasc Biol. 2003.23: 853-858). Studies also demonstrate that ApoCIII is a key determinantin the clearance of triglyceride-rich lipoproteins and its remnants inhypertriglyceridaemic states, including visceral obesity, insulinresistance and the metabolic syndrome (Mauger et al., J. Lipid Res.2006. 47: 1212-1218; Chan et al., Clin. Chem. 2002. 278-283; Ooi et al.,Clin. Sci. 2008. 114: 611-624).

Hypertriglyceridemia is a common clinical trait associated with anincreased risk of cardiometabolic disease (Hegele et al. 2009, Hum MolGenet, 18: 4189-4194; Hegele and Pollex 2009, Mol Cell Biochem, 326:35-43) as well as of occurrence of acute pancreatitis in the most severeforms (Toskes 1990, Gastroenterol Clin North Am, 19: 783-791; Gaudet etal. 2010, Atherosclerosis Supplements, 11: 55-60; Catapano et al. 2011,Atherosclerosis, 217S: S1-S44; Tremblay et al. 2011, J Clin Lipidol, 5:37-44). Examples of cardiometabolic disease include, but are not limitedto, diabetes, metabolic syndrome/insulin resistance, and geneticdisorders such as familial chylomicronemia, familial combinedhyperlipidemia and familial hypertriglyceridemia.

Hypertriglyceridemia is the consequence of increased production and/orreduced or delayed catabolism of triglyceride (TG)-rich lipoproteins:VLDL and, to a lesser extent, chylomicrons (CM). Borderline high TGs(150-199 mg/dL) are commonly found in the general population and are acommon component of the metabolic syndrome/insulin resistance states.The same is true for high TGs (200-499 mg/dL) except that as plasma TGlevels increase, underlying genetic factors play an increasinglyimportant etiologic role. Very high TGs (≧500 mg/dL) are most oftenassociated with elevated CM levels as well, and are accompanied byincreasing risk for acute pancreatitis. The risk of pancreatitis isconsidered clinically significant if TG exceeds 880 mg/dL (>10 mmol) andthe European Atherosclerosis Society/European Society of Cardiology(EAS/ESC) 2011 guidelines state that actions to prevent acutepancreatitis are mandatory (Catapano et al. 2011, Atherosclerosis, 217S:S1-S44). According to the EAS/ESC 2011 guidelines, hypertriglyceridemiais the cause of approximately 10% of all cases of pancreatitis, anddevelopment of pancreatitis can occur at TG levels between 440-880mg/dL. Based on evidence from clinical studies demonstrating thatelevated TG levels are an independent risk factor for atheroscleroticCVD, the guidelines from both the National Cholesterol Education ProgramAdult Treatment Panel III (NCEP 2002, Circulation, 106: 3143-421) andthe American Diabetes Association (ADA 2008, Diabetes Care, 31:S12-S54.) recommend a target TG level of less than 150 mg/dL to reducecardiovascular risk.

ApoCIII-knockout mice had normal intestinal lipid absorption and hepaticVLDL triacylglycerol secretion, but a rapid clearance of VLDLtriacylglycerols and VLDL cholesteryl esters from plasma that mayexplain the observed hypolipidaemia (Gerritsen et al., J. Lipid Res.2005. 46: 1466-1473; Jong et al., J. Lipid Res. 2001. 42: 1578-1585).VLDL particles with ApoCIII have been cited to play a major role inidentifying the high risk of coronary heart disease inhypertriglyceridemia (Campos et al., J. Lipid Res. 2001. 42: 1239-1249).A genome-wide association study found a naturally occurring ApoCIII nullmutation in Lancaster Amish people demonstrated a favorable lipidprofile and apparent cardioprotection, with no obvious detrimentaleffects (Pollin et al., Science. 2008. 322: 1702-1705). The mutationcarriers are observed to have lower fasting and postprandial serumtriglycerides and LDL-cholesterol, and higher levels of HDL-cholesterol.

The HDL class of lipoproteins comprises a heterogeneous and polydispersepopulation of particles that are the most dense and smallest of size(Havel and Kane. In, The Metabolic & Molecular Bases of InheritedDisease. 8^(th) Edition. McGraw-Hill, New York, 2001:2705-16). HDL is amacromolecular complex of lipids (cholesterol, triglycerides andphospholipids) and proteins (apolipoproteins (apo) and enzymes). Thesurface of HDL contains chiefly apolipoproteins A, C and E. The functionof some of these apoproteins is to direct HDL from the peripheraltissues to the liver. Serum HDL levels can be affected by underlyinggenetic causes (Weissglas-Volkov and Pajukanta, J Lipid Res, 2010,51:2032-2057).

Epidemiological studies have indicated that increased levels of HDLprotect against cardiovascular disease or coronary heart disease (Gordonet al., Am. J. Med. 1977. 62: 707-714). These effects of HDL-cholesterolare independent of triglyceride and LDL-cholesterol concentrations. Inclinical practice, a low plasma HDL-cholesterol is more commonlyassociated with other disorders that increase plasma triglycerides, forexample, central obesity, insulin resistance, type 2 diabetes mellitusand renal disease (chronic renal failure or nephrotic proteinuria)(Kashyap. Am. J. Cardiol. 1998. 82: 42U-48U).

Currently, there are no known direct therapeutic agents that affect thefunction of ApoCIII. The hypolipidemic effect of the fibrate class ofdrugs has been postulated to occur via a mechanism where peroxisomeproliferator activated receptor (PPAR) mediates the displacement ofHNF-4 from the apolipoprotein C-III promoter, resulting intranscriptional suppression of apolipoprotein C-III (Hertz et al., J.Biol. Chem., 1995, 270, 13470-13475). The statin class of hypolipidemicdrugs also lower triglyceride levels via an unknown mechanism, whichresults in increases in lipoprotein lipase mRNA and a decrease in plasmalevels of apolipoprotein C-III (Schoonjans et al., FEBS Lett., 1999,452, 160-164). Consequently, there remains a long felt need foradditional agents capable of effectively inhibiting apolipoprotein C-IIIfunction.

Antisense technology is emerging as an effective means for reducing theexpression of certain gene products and may therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications for the modulation of ApoCIII.

We have previously disclosed compositions and method for inhibitingApoCIII by antisense compounds in US 20040208856 (U.S. Pat. No.7,598,227), US 20060264395 (U.S. Pat. No. 7,750,141), and WO2004/093783. In the present application, we disclose the unexpectedresult that antisense inhibition of ApoCIII resulted in the elevation ofHDL levels and decrease in postprandial triglyceride levels. This resultwill be useful, for example, to treat, prevent, delay, decrease orameliorate any one or more diseases, such as cardiovascular disease(e.g., coronary heart disease or atherogenic diseases). For example,elevated postprandial (non-fasting) triglyceride levels have beenidentified as a significant risk factor for cardiovascular diseases(Bansal et al., JAMA, 2007, 298:309-16; Nordestgaard et al., JAMA, 2007,298:299-308), Also, in the present application, inhibition of ApoCIIIexpression unexpectedly results in increased chylomicron clearance andis therefore important in the prevention of chylomicronemia (Chait etal., 1992, Adv Intern Med. 1992, 37:249-73), a dyslipidemic state causedby improper clearance of chylomicron triglyceride. Severe forms ofchylomicronemia can lead to pancreatitis, a life-threatening condition.By inhibiting intestinal ApoCIII, inhibition of lipoprotein lipase wouldbe reduced, and chylomicron triglyceride clearance would be enhanced,thereby preventing pancreatitis.

SUMMARY OF THE INVENTION

Provided herein are methods of increasing HDL levels by administering toan animal a compound targeting ApoCIII.

Certain embodiments provide a method of preventing, treating,ameliorating, delaying the onset of or reducing the risk of acardiovascular disease, disorder or condition in an animal comprisingadministering a compound targeting ApoCIII to the animal. The compoundadministered to the animal prevents, treats, ameliorates, delays theonset of, or reduces the risk of, the cardiovascular disease, disorderor condition by increasing HDL levels in the animal.

Certain embodiments provide a method of reducing the risk for acardiovascular disease in an animal comprising administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides,wherein the modified oligonucleotide is complementary to an ApoCIIInucleic acid. In certain embodiments, the ApoCIII nucleic acid is asshown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, thecompound comprises a modified oligonucleotide consisting of 12 to 30linked nucleosides, and having a nucleobase sequence comprising at least8 contiguous nucleobases of ISIS 304801 (SEQ ID NO: 3). In furtherembodiments, the compound administered to the animal reduces the riskfor a cardiovascular disease, by increasing HDL levels.

Certain embodiments provide a method of preventing, treating,ameliorating or reducing at least one symptom of a cardiovasculardisease in an animal, comprising administering to the animal atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides, wherein themodified oligonucleotide is complementary to an ApoCIII nucleic acid. Incertain embodiments, the ApoCIII nucleic acid is as shown in SEQ ID NO:1 or SEQ ID NO: 2. In certain embodiments, the compound comprises amodified oligonucleotide consisting of 12 to 30 linked nucleosides, andhaving a nucleobase sequence comprising at least 8 contiguousnucleobases of ISIS 304801 (SEQ ID NO: 3). In further embodiments, thecompound administered to the animal prevents, treats, ameliorates orreduces at least one symptom of the cardiovascular disease in theanimal, by increasing HDL levels in the animal.

Certain embodiments provide a method of raising HDL levels in an animalby administering to the animal a compound consisting of ISIS 304801 (SEQID NO: 3) to raise the HDL levels in the animal.

Certain embodiments provide a method of preventing, treating,ameliorating or reducing at least one symptom of a cardiovasculardisease in an animal by administering to the animal a compoundconsisting of ISIS 304801 (SEQ ID NO: 3) to prevent, treat, ameliorateor reduce at least one symptom of the cardiovascular disease in theanimal, by increasing HDL levels in the animal.

Certain embodiments provide a method of raising HDL levels in an animalby administering to the animal a modified oligonucleotide, having thesequence of SEQ ID NO: 3 (ISIS 304801) wherein the modifiedoligonucleotide comprises: a gap segment consisting of 10 linkeddeoxynucleosides; a 5′ wing segment consisting of 5 linked nucleosides;a 3′ wing segment consisting 5 linked nucleosides; wherein the gapsegment is positioned immediately adjacent to and between the 5′ wingsegment and the 3′ wing segment and wherein each nucleoside of each wingsegment comprises a 2′-O-methyoxyethyl sugar, wherein each cytosine is a5′-methylcytosine, and wherein each internucleoside linkage is aphosphorothioate linkage, wherein the modified oligonucleotide raisesthe HDL levels in the animal.

Certain embodiments provide a method of preventing, treating,ameliorating or reducing at least one symptom of a cardiovasculardisease in an animal by administering to the animal a modifiedoligonucleotide, having the sequence of ISIS 304801 (SEQ ID NO: 3)wherein the modified oligonucleotide comprises: a gap segment consistingof 10 linked deoxynucleosides; a 5′ wing segment consisting of 5 linkednucleosides; a 3′ wing segment consisting 5 linked nucleosides; whereinthe gap segment is positioned immediately adjacent to and between the 5′wing segment and the 3′ wing segment and wherein each nucleoside of eachwing segment comprises a 2′-O-methyoxyethyl sugar, wherein each cytosineis a 5′-methylcytosine, and wherein each internucleoside linkage is aphosphorothioate linkage, wherein the modified oligonucleotide prevents,treats, ameliorates or reduces at least one symptom in the animal withthe cardiovascular disease by raising the HDL levels in the animal.

Certain embodiments provide a method of raising HDL levels in an animalby administering to the animal a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides, wherein themodified oligonucleotide is complementary to an ApoCIII nucleic acid, asshown in SEQ ID NO: 1 or SEQ ID NO: 2, to raise the HDL levels in theanimal.

Certain embodiments provide a method of preventing, treating,ameliorating or reducing at least one symptom of a cardiovasculardisease in an animal by administering to the animal a compoundcomprising a modified oligonucleotide consisting of 12 to 30 linkednucleosides, wherein the modified oligonucleotide is complementary to anApoCIII nucleic acid, as shown in SEQ ID NO: 1 or SEQ ID NO: 2, topreventing, treating, ameliorating or reducing at least one symptom ofthe cardiovascular disease in the animal, by raising the HDL levels ofthe animal.

Certain embodiments provide a method of decreasing CETP levels byadministering a compound targeting ApoCIII to an animal. In certainembodiments, the compound comprises a modified oligonucleotideconsisting of 12 to 30 linked nucleosides, and having a nucleobasesequence complementary to an ApoCIII nucleic acid. In certainembodiments, the nucleobase sequence comprises at least 8 contiguousnucleobases of ISIS 304801 (SEQ ID NO: 3). In certain embodiments, thecompound consists of the nucleobases of ISIS 304801 (SEQ ID NO: 3).

Certain embodiments provide a method of increasing ApoA1, PON1, fatclearance, chylomicron triglyceride clearance, post prandialtriglyceride clearance or HDL by administering a compound targetingApoCIII to an animal. In certain embodiments, the compound comprises amodified oligonucleotide consisting of 12 to 30 linked nucleosides, andhaving a nucleobase sequence complementary to an ApoCIII nucleic acid.In certain embodiments, the nucleobase sequence comprises at least 8contiguous nucleobases of ISIS 304801 (SEQ ID NO: 3). In certainembodiments, the compound consists of the nucleobases of ISIS 304801(SEQ ID NO: 3).

Certain embodiments provide a method of preventing, delaying orameliorating pancreatitis comprising: (a) selecting an animal with, orat risk of, pancreatitis, and (b) administering a compound targetingApoCIII to the animal, wherein the pancreatitis is prevented, delayed orameliorated.

Certain embodiments provide a method of preventing, delaying orameliorating pancreatitis comprising: (a) selecting an animal with, orat risk of, pancreatiis, and (b) administering a compound targetingApoCIII to the animal, thereby increasing chylomicron clearance, whereinthe pancreatitis is prevented, delayed or ameliorated.

In certain embodiments, the animal has, or is at risk for,hypertriglyceridemia. In certain embodiments, the hypertriglyceridemiais Fredrickson Type II, IV or V. In certain embodiments, the animal hasa genetic defect leading to hypertriglyceridemia. In certainembodiments, the genetic defect is a heterozygous LPL deficiency or anApoCIII polymorphism. In certain embodiments, the animal has atriglyceride level ≧500 mg/dL and a heterozygous LPL deficiency.

In certain embodiments, the animal has a triglyceride level between100-200 mg/dL, 100-300 mg/dL, 100-400 mg/dL, 100-500 mg/dL, 200-500mg/dL, 300-500 mg/dL, 400-500 mg/dL, 500-1000 mg/dL, 600-1000 mg/dL,700-1000 mg/dL, 800-1000 mg/dL, 900-1000 mg/dL, 500-1500 mg/dL,1000-1500 mg/dL, 100-2000 mg/dL, 150-2000 mg/dL, 200-2000 mg/dL,300-2000 mg/dL, 400-2000 mg/dL, 500-2000 mg/dL, 600-2000 mg/dL, 700-2000mg/dL, 800-2000 mg/dL, 900-2000 mg/dL, 1000-2000 mg/dL, 1100-2000 mg/dL,1200-2000 mg/dL, 1300-2000 mg/dL, 1400-2000 mg/dL, or 1500-2000 mg/dL.

In certain embodiments, increased chylomicron clearance enhancesclearance of postprandial triglycerides and/or decreases postprandialtriglycerides.

Certain embodiments provide a use of a compound targeted to ApoCIII forpreventing, treating, ameliorating or reducing at least one symptom of acardiovascular disease, by increasing HDL levels.

Certain embodiments provide a use of a compound targeted to ApoCIII forincreasing HDL levels in an animal.

Certain embodiments provide a use of a compound targeted to ApoCIII forthe preparation of a medicament for increasing HDL levels in an animal.

Certain embodiments provide a use of a compound targeted to ApoCIII forthe preparation of a medicament for improving the ratio of TG to HDL.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference forthe portions of the document discussed herein, as well as in theirentirety.

DEFINITIONS

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Where permitted, all patents, applications, publishedapplications and other publications, GENBANK Accession Numbers andassociated sequence information obtainable through databases such asNational Center for Biotechnology Information (NCBI) and other datareferred to throughout in the disclosure herein are incorporated byreference for the portions of the document discussed herein, as well asin their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

“2′-O-methoxyethyl” (also 2′-MOE, 2′-O(CH₂)₂—OCH₃ and2′-O-(2-methoxyethyl)) refers to an O-methoxy-ethyl modification of the2′ position of a furosyl ring. A 2′-O-methoxyethyl modified sugar is amodified sugar.

“2′-O-methoxyethyl nucleotide” means a nucleotide comprising a2′-O-methoxyethyl modified sugar moiety.

“3′ target site” refers to the nucleotide of a target nucleic acid whichis complementary to the 3′-most nucleotide of a particular antisensecompound.

“5′ target site” refers to the nucleotide of a target nucleic acid whichis complementary to the 5′-most nucleotide of a particular antisensecompound.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5′ position. A 5-methylcytosine is a modifiednucleobase.

“About” means within ±10% of a value. For example, if it is stated, “amarker may be increased by about 50%”, it is implied that the marker maybe increased between 45%-55%.

“Active pharmaceutical agent” means the substance or substances in apharmaceutical composition that provide a therapeutic benefit whenadministered to an individual. For example, in certain embodiments anantisense oligonucleotide targeted to ApoCIII is an activepharmaceutical agent.

“Active target region” or “target region” means a region to which one ormore active antisense compounds is targeted. “Active antisensecompounds” means antisense compounds that reduce target nucleic acidlevels or protein levels.

“Administered concomitantly” refers to the co-administration of twoagents in any manner in which the pharmacological effects of both aremanifest in the patient at the same time. Concomitant administrationdoes not require that both agents be administered in a singlepharmaceutical composition, in the same dosage form, or by the sameroute of administration. The effects of both agents need not manifestthemselves at the same time. The effects need only be overlapping for aperiod of time and need not be coextensive.

“Administering” means providing a pharmaceutical agent to an individual,and includes, but is not limited to administering by a medicalprofessional and self-administering.

“Agent” means an active substance that can provide a therapeutic benefitwhen administered to an animal. “First Agent” means a therapeuticcompound of the invention. For example, a first agent can be anantisense oligonucleotide targeting ApoCIII. “Second agent” means asecond therapeutic compound of the invention (e.g. a second antisenseoligonucleotide targeting ApoCIII) and/or a non-ApoCIII therapeuticcompound.

“Amelioration” refers to a lessening of at least one indicator, sign, orsymptom of an associated disease, disorder, or condition. The severityof indicators may be determined by subjective or objective measures,which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but notlimited to, mice, rats, rabbits, dogs, cats, pigs, and non-humanprimates, including, but not limited to, monkeys and chimpanzees.

“Antisense activity” means any detectable or measurable activityattributable to the hybridization of an antisense compound to its targetnucleic acid. In certain embodiments, antisense activity is a decreasein the amount or expression of a target nucleic acid or protein encodedby such target nucleic acid.

“Antisense compound” means an oligomeric compound that is capable ofundergoing hybridization to a target nucleic acid through hydrogenbonding. As used herein, the term “antisense compound” encompassespharmaceutically acceptable derivatives of the compounds describedherein.

“Antisense inhibition” means the reduction of target nucleic acid levelsor target protein levels in the presence of an antisense compoundcomplementary to a target nucleic acid compared to target nucleic acidlevels or target protein levels in the absence of the antisensecompound.

“Antisense oligonucleotide” means a single-stranded oligonucleotidehaving a nucleobase sequence that permits hybridization to acorresponding region or segment of a target nucleic acid. As usedherein, the term “antisense oligonucleotide” encompassespharmaceutically acceptable derivatives of the compounds describedherein.

“ApoCIII” means any nucleic acid or protein sequence encoding ApoCIII.For example, in certain embodiments, an ApoCIII includes a DNA sequenceencoding ApoCIII, a RNA sequence transcribed from DNA encoding ApoCIII(including genomic DNA comprising introns and exons), a mRNA sequenceencoding ApoCIII, or a peptide sequence encoding ApoCIII.

“ApoCIII mRNA” means a mRNA encoding an ApoCIII protein.

“ApoCIII protein” means any protein sequence encoding ApoCIII.

“Atherosclerosis” means a hardening of the arteries affecting large andmedium-sized arteries and is characterized by the presence of fattydeposits. The fatty deposits are called “atheromas” or “plaques,” whichconsist mainly of cholesterol and other fats, calcium and scar tissue,and damage the lining of arteries.

“Bicyclic sugar” means a furosyl ring modified by the bridging of twonon-geminal ring atoms. A bicyclic sugar is a modified sugar.

“Bicyclic nucleic acid” or “BNA” refers to a nucleoside or nucleotidewherein the furanose portion of the nucleoside or nucleotide includes abridge connecting two carbon atoms on the furanose ring, thereby forminga bicyclic ring system.

“Cap structure” or “terminal cap moiety” means chemical modifications,which have been incorporated at either terminus of an antisensecompound.

“Cardiovascular disease” or “cardiovascular disorder” refers to a groupof conditions related to the heart, blood vessels, or the circulation.Examples of cardiovascular diseases include, but are not limited to,aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular disease(stroke), coronary heart disease, hypertension, dyslipidemia,hyperlipidemia, hypertriglyceridemia and hypercholesterolemia.

“Chemically distinct region” refers to a region of an antisense compoundthat is in some way chemically different than another region of the sameantisense compound. For example, a region having 2′-O-methoxyethylnucleotides is chemically distinct from a region having nucleotideswithout 2′-O-methoxyethyl modifications.

“Chimeric antisense compound” means an antisense compound that has atleast two chemically distinct regions.

“Cholesterol” is a sterol molecule found in the cell membranes of allanimal tissues. Cholesterol must be transported in an animal's bloodplasma by lipoproteins including very low density lipoprotein (VLDL),intermediate density lipoprotein (IDL), low density lipoprotein (LDL),and high density lipoprotein (HDL). “Plasma cholesterol” refers to thesum of all lipoproteins (VDL, IDL, LDL, HDL) esterified and/ornon-esterified cholesterol present in the plasma or serum.

“Cholesterol absorption inhibitor” means an agent that inhibits theabsorption of exogenous cholesterol obtained from diet.

“Co-administration” means administration of two or more agents to anindividual. The two or more agents can be in a single pharmaceuticalcomposition, or can be in separate pharmaceutical compositions. Each ofthe two or more agents can be administered through the same or differentroutes of administration. Co-administration encompasses parallel orsequential administration.

“Complementarity” means the capacity for pairing between nucleobases ofa first nucleic acid and a second nucleic acid. In certain embodiments,complementarity between the first and second nucleic acid can be betweentwo DNA strands, between two RNA strands, or between a DNA and an RNAstrand. In certain embodiments, some of the nucleobases on one strandare matched to a complementary hydrogen bonding base on the otherstrand. In certain embodiments, all of the nucleobases on one strand arematched to a complementary hydrogen bonding base on the other strand. Incertain embodiments, a first nucleic acid is an antisense compound and asecond nucleic acid is a target nucleic acid. In certain suchembodiments, an antisense oligonucleotide is a first nucleic acid and atarget nucleic acid is a second nucleic acid.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother.

“Constrained ethyl” or “cEt” refers to a bicyclic nucleoside having afuranosyl sugar that comprises a methyl(methyleneoxy) (4′-CH(CH₃)—O-2′)bridge between the 4′ and the 2′ carbon atoms.

“Cross-reactive” means an oligomeric compound targeting one nucleic acidsequence can hybridize to a different nucleic acid sequence. Forexample, in some instances an antisense oligonucleotide targeting humanApoCIII can cross-react with a murine ApoCIII. Whether an oligomericcompound cross-reacts with a nucleic acid sequence other than itsdesignated target depends on the degree of complementarity the compoundhas with the non-target nucleic acid sequence. The higher thecomplementarity between the oligomeric compound and the non-targetnucleic acid, the more likely the oligomeric compound will cross-reactwith the nucleic acid.

“Cure” means a method that restores health or a prescribed treatment foran illness.

“Coronary heart disease (CHD)” means a narrowing of the small bloodvessels that supply blood and oxygen to the heart, which is often aresult of atherosclerosis.

“Deoxyribonucleotide” means a nucleotide having a hydrogen at the 2′position of the sugar portion of the nucleotide. Deoxyribonucleotidesmay be modified with any of a variety of substituents.

“Diabetes mellitus” or “diabetes” is a syndrome characterized bydisordered metabolism and abnormally high blood sugar (hyperglycemia)resulting from insufficient levels of insulin or reduced insulinsensitivity. The characteristic symptoms are excessive urine production(polyuria) due to high blood glucose levels, excessive thirst andincreased fluid intake (polydipsia) attempting to compensate forincreased urination, blurred vision due to high blood glucose effects onthe eye's optics, unexplained weight loss, and lethargy.

“Diabetic dyslipidemia” or “type 2 diabetes with dyslipidemia” means acondition characterized by Type 2 diabetes, reduced HDL-C, elevatedtriglycerides, and elevated small, dense LDL particles.

“Diluent” means an ingredient in a composition that lackspharmacological activity, but is pharmaceutically necessary ordesirable. For example, the diluent in an injected composition may be aliquid, e.g. saline solution.

“Dyslipidemia” refers to a disorder of lipid and/or lipoproteinmetabolism, including lipid and/or lipoprotein overproduction ordeficiency. Dyslipidemias may be manifested by elevation of lipids suchas chylomicron, cholesterol and triglycerides as well as lipoproteinssuch as low-density lipoprotein (LDL) cholesterol. An example of adyslipidemia is chylomicronemia.

“Dosage unit” means a form in which a pharmaceutical agent is provided,e.g. pill, tablet, or other dosage unit known in the art. In certainembodiments, a dosage unit is a vial containing lyophilized antisenseoligonucleotide. In certain embodiments, a dosage unit is a vialcontaining reconstituted antisense oligonucleotide.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration, or in a specified time period. In certainembodiments, a dose can be administered in one, two, or more boluses,tablets, or injections. For example, in certain embodiments wheresubcutaneous administration is desired, the desired dose requires avolume not easily accommodated by a single injection, therefore, two ormore injections can be used to achieve the desired dose. In certainembodiments, the pharmaceutical agent is administered by infusion overan extended period of time or continuously. Doses can be stated as theamount of pharmaceutical agent per hour, day, week, or month. Doses canalso be stated as mg/kg or g/kg.

“Effective amount” or “therapeutically effective amount” means theamount of active pharmaceutical agent sufficient to effectuate a desiredphysiological outcome in an individual in need of the agent. Theeffective amount can vary among individuals depending on the health andphysical condition of the individual to be treated, the taxonomic groupof the individuals to be treated, the formulation of the composition,assessment of the individual's medical condition, and other relevantfactors.

The “Fredrickson” system is used to classify primary (genetic) causes ofdislipidemia into several subgroups or types. Dislipidemia types thatmay be amenable to therapy with the compounds disclosed herein include,but are not limited to, Fredrickson Type II, IV and V.

“Fredrickson Type I” exists in several forms: Type 1a is a lipoproteinlipase deficiency due to a deficiency of LPL or altered apoC-II; Type Ibis a familial apoprotein CII deficiency, a condition caused by a lack oflipoprotein lipase activator; and Type Ic is a chylomicronemia due tocirculating inhibitor of lipoprotein lipase. Type I is a rare disorderthat usually presents in childhood. It is characterized by severeelevations in chylomicrons and extremely elevated TG levels (alwaysreaching well above 1000 mg/dL and not infrequently rising as high as10,000 mg/dL or more) with episodes of abdominal pain, recurrent acutepancreatitis, eruptive cutaneous xanthomata, and hepatosplenomegaly.Patients rarely develop atherosclerosis, perhaps because their plasmalipoprotein particles are too large to enter into the arterial intima(Nordestgaard et al., J Lipid Res, 1988, 29:1491-1500; Nordestgaard etal., Arteriosclerosis, 1988, 8:421-428). Type I is usually caused bymutations of either the LPL gene, or of the gene's cofactor apoC-II,resulting in the inability of affected individuals to producefunctionally active LPL. Patients are either homozygous for suchmutations or compound heterozygous. The prevalence is approximately 1 in1,000,000 in the general population and much higher in South Africa andEastern Quebec as a result of a founder effect. Patients respondminimally, or not at all, to TG-lowering drugs (Tremblay et al., J ClinLipidol, 2011, 5:37-44; Brisson et al., Pharmacogenet Genom, 2010,20:742-747) and hence restriction of dietary fat to 20 grams/day or lessis used to manage the symptoms of this rare disorder.

“Fredrickson Type II” is the most common form of primary hyperlipidemia.It is further classified into Type IIa and Type IIb, depending mainly onwhether there is elevation in VLDL in addition to LDL cholesterol(LDL-C). Type IIa (familial hypercholesterolemia) may be sporadic (dueto dietary factors), polygenic, or truly familial as a result of amutation in either the LDL receptor gene on chromosome 19 (0.2% of thepopulation) or the apolipoprotein B (apoB) gene (0.2%). The familialform is characterized by tendon xanthoma, xanthelasma and prematurecardiovascular disease. The incidence of this disease is about 1 in 500for heterozygotes, and 1 in 1,000,000 for homozygotes. Type IIb (alsoknown as familial combined hyperlipoproteinemia) is a mixedhyperlipidemia (high cholesterol and TG levels), caused by elevations inLDL-C and in VLDL. The high VLDL levels are due to overproduction ofsubstrates, including TG, acetyl CoA, and an increase in B-100synthesis. They may also be caused by the decreased clearance of LDL.Prevalence in the population is about 10%.

“Fredrickson Type III” (also known as dysbetalipoproteinemia) is aremnant removal disease, or broad-beta disease (Fern et al., J ClinPathol, 2008, 61:1174-118). It is due to cholesterol-rich VLDL (β-VLDL).Typically, patients with this condition have elevated plasma cholesteroland TG levels because of impaired clearance of chylomicron and VLDLremnants (e.g. IDL). The impaired clearance is due to a defect inapolipoprotein E (apoE). Normally functioning apoE contained on theremnants would enable binding to the LDL receptor and removal from thecirculation. Accumulation of the remnants in affected individuals canresult in xanthomatosis and premature coronary and/or peripheralvascular disease. The most common cause for Type III is the presence ofapoE E2/E2 genotype. Its prevalence has been estimated to beapproximately 1 in 10,000.

“Fredrickson Type IV” (also known as familial hypertriglyceridemia) isan autosomal dominant condition occurring in approximately 1% of thepopulation. TG levels are elevated as a result of excess hepaticproduction of VLDL or heterozygous LPL deficiency, but are almost alwaysless than 1000 mg/dL. Serum cholesterol levels are usually within normallimits. The disorder is heterogeneous and the phenotype stronglyinfluenced by environmental factors, particularly carbohydrate andethanol consumption.

“Fredrickson Type V” has high VLDL and chylomicrons. It is characterizedby carriers of loss-of-function LPL gene variants associated with LPLactivity of at least 20% (i.e. partial LPL deficiency as compared toFredrickson Type I). These patients present with lactescent plasma andsevere hypertriglyceridemia because of chylomicrons and VLDL. TG levelsare invariably greater than 1000 mg/dL and total cholesterol levels arealways elevated. The LDL-C level is usually low. It is also associatedwith increased risk for acute pancreatitis, glucose intolerance andhyperuricemia. Symptoms generally present in adulthood (>35 years) and,although the prevalence is relatively rare, it is much more common thanhomozygous or compound heterozygous LPL deficient patients.

“Fully complementary” or “100% complementary” means each nucleobase of anucleobase sequence of a first nucleic acid has a complementarynucleobase in a second nucleobase sequence of a second nucleic acid. Incertain embodiments, a first nucleic acid is an antisense compound and asecond nucleic acid is a target nucleic acid.

“Gapmer” means a chimeric antisense compound in which an internal regionhaving a plurality of nucleosides that support RNase H cleavage ispositioned between external regions having one or more nucleosides,wherein the nucleosides comprising the internal region are chemicallydistinct from the nucleoside or nucleosides comprising the externalregions. The internal region may be referred to as a “gap” or “gapsegment” and the external regions may be referred to as “wings” or “wingsegments.”

“Gap-widened” means a chimeric antisense compound having a gap segmentof 12 or more contiguous 2′-deoxyribonucleosides positioned between andimmediately adjacent to 5′ and 3′ wing segments having from one to sixnucleosides.

“Glucose” is a monosaccharide used by cells as a source of energy andinflammatory intermediate. “Plasma glucose” refers to glucose present inthe plasma.

“High density lipoprotein-C(HDL-C)” means cholesterol associated withhigh density lipoprotein particles. Concentration of HDL-C in serum (orplasma) is typically quantified in mg/dL or nmol/L. “HDL-C” and “plasmaHDL-C” mean HDL-C in serum and plasma, respectively.

“HMG-CoA reductase inhibitor” means an agent that acts through theinhibition of the enzyme HMG-CoA reductase, such as atorvastatin,rosuvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin.

“Hybridization” means the annealing of complementary nucleic acidmolecules. In certain embodiments, complementary nucleic acid moleculesinclude an antisense compound and a target nucleic acid.

“Hypercholesterolemia” means a condition characterized by elevatedcholesterol or circulating (plasma) cholesterol, LDL-cholesterol andVLDL-cholesterol, as per the guidelines of the Expert Panel Report ofthe National Cholesterol Educational Program (NCEP) of Detection,Evaluation of Treatment of high cholesterol in adults (see, Arch. Int.Med. (1988) 148, 36-39).

“Hyperlipidemia” or “hyperlipemia” is a condition characterized byelevated serum lipids or circulating (plasma) lipids. This conditionmanifests an abnormally high concentration of fats. The lipid fractionsin the circulating blood are cholesterol, low density lipoproteins, verylow density lipoproteins, chylomicrons and triglycerides. TheFredrickson classification of hyperlipidemias is based on the pattern ofTG and cholesterol-rich lipoprotein particles, as measured byelectrophoresis or ultracentrifugation and is commonly used tocharacterize primary causes of hyperlipidemias such ashypertriglyceridemia (Fredrickson and Lee, Circulation, 1965,31:321-327; Fredrickson et al., New Eng J Med, 1967, 276 (1): 34-42).

“Hypertriglyceridemia” means a condition characterized by elevatedtriglyceride levels. Its etiology includes primary (i.e. genetic causes)and secondary (other underlying causes such as diabetes, metabolicsyndrome/insulin resistance, obesity, physical inactivity, cigarettesmoking, excess alcohol and a diet very high in carbohydrates) factorsor, most often, a combination of both (Yuan et al. CMAJ, 2007,176:1113-1120).

“Identifying” or “selecting an animal with metabolic or cardiovasculardisease” means identifying or selecting a subject prone to or havingbeen diagnosed with a metabolic disease, a cardiovascular disease, or ametabolic syndrome; or, identifying or selecting a subject having anysymptom of a metabolic disease, cardiovascular disease, or metabolicsyndrome including, but not limited to, hypercholesterolemia,hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypertensionincreased insulin resistance, decreased insulin sensitivity, abovenormal body weight, and/or above normal body fat content or anycombination thereof. Such identification can be accomplished by anymethod, including but not limited to, standard clinical tests orassessments, such as measuring serum or circulating (plasma)cholesterol, measuring serum or circulating (plasma) blood-glucose,measuring serum or circulating (plasma) triglycerides, measuringblood-pressure, measuring body fat content, measuring body weight, andthe like.

“Improved cardiovascular outcome” means a reduction in the occurrence ofadverse cardiovascular events, or the risk thereof. Examples of adversecardiovascular events include, without limitation, death, reinfarction,stroke, cardiogenic shock, pulmonary edema, cardiac arrest, and atrialdysrhythmia.

“Immediately adjacent” means there are no intervening elements betweenthe immediately adjacent elements, for example, between regions,segments, nucleotides and/or nucleosides.

“Increasing HDL” or “raising HDL” means increasing the level of HDL inan animal after administration of at least one compound of theinvention, compared to the HDL level in an animal not administered anycompound.

“Individual” or “subject” or “animal” means a human or non-human animalselected for treatment or therapy.

“Induce”, “inhibit”, “potentiate”, “elevate”, “increase”, “decrease”,“reduce” or the like denote quantitative differences between two states.For example, “an amount effective to inhibit the activity or expressionof ApoCIII” means that the level of activity or expression of ApoCIII ina treated sample will differ from the level of ApoCIII activity orexpression in an untreated sample. Such terms are applied to, forexample, levels of expression, and levels of activity.

“Inhibiting the expression or activity” refers to a reduction orblockade of the expression or activity of a RNA or protein and does notnecessarily indicate a total elimination of expression or activity.

“Insulin resistance” is defined as the condition in which normal amountsof insulin are inadequate to produce a normal insulin response from fat,muscle and liver cells. Insulin resistance in fat cells results inhydrolysis of stored triglycerides, which elevates free fatty acids inthe blood plasma. Insulin resistance in muscle reduces glucose uptakewhereas insulin resistance in liver reduces glucose storage, with botheffects serving to elevate blood glucose. High plasma levels of insulinand glucose due to insulin resistance often leads to metabolic syndromeand type 2 diabetes.

“Insulin sensitivity” is a measure of how effectively an individualprocesses glucose. An individual having high insulin sensitivityeffectively processes glucose whereas an individual with low insulinsensitivity does not effectively process glucose.

“Internucleoside linkage” refers to the chemical bond betweennucleosides.

“Intravenous administration” means administration into a vein.

“Linked nucleosides” means adjacent nucleosides which are bondedtogether.

“Lipid-lowering” means a reduction in one or more lipids in a subject.“Lipid-raising” means an increase in a lipid (e.g., HDL) in a subject.Lipid-lowering or lipid-raising can occur with one or more doses overtime.

“Lipid-lowering therapy” or “lipid lowering agent” means a therapeuticregimen provided to a subject to reduce one or more lipids in a subject.In certain embodiments, a lipid-lowering therapy is provided to reduceone or more of CETP, ApoB, total cholesterol, LDL-C, VLDL-C, IDL-C,non-HDL-C, triglycerides, small dense LDL particles, and Lp(a) in asubject. Examples of lipid-lowering therapy include statins, fibrates,MTP inhibitors.

“Lipoprotein”, such as VLDL, LDL and HDL, refers to a group of proteinsfound in the serum, plasma and lymph and are important for lipidtransport. The chemical composition of each lipoprotein differs in thatthe HDL has a higher proportion of protein versus lipid, whereas theVLDL has a lower proportion of protein versus lipid.

“Low density lipoprotein-cholesterol (LDL-C)” means cholesterol carriedin low density lipoprotein particles. Concentration of LDL-C in serum(or plasma) is typically quantified in mg/dL or nmol/L. “Serum LDL-C”and “plasma LDL-C” mean LDL-C in the serum and plasma, respectively.

“Major risk factors” refers to factors that contribute to a high riskfor a particular disease or condition. In certain embodiments, majorrisk factors for coronary heart disease include, without limitation,cigarette smoking, hypertension, low HDL-C, family history of coronaryheart disease, age, and other factors disclosed herein.

“Metabolic disorder” or “metabolic disease” refers to a conditioncharacterized by an alteration or disturbance in metabolic function.“Metabolic” and “metabolism” are terms well known in the art andgenerally include the whole range of biochemical processes that occurwithin a living organism. Metabolic disorders include, but are notlimited to, hyperglycemia, prediabetes, diabetes (type 1 and type 2),obesity, insulin resistance, metabolic syndrome and dyslipidemia due totype 2 diabetes.

“Metabolic syndrome” means a condition characterized by a clustering oflipid and non-lipid cardiovascular risk factors of metabolic origin. Incertain embodiments, metabolic syndrome is identified by the presence ofany 3 of the following factors: waist circumference of greater than 102cm in men or greater than 88 cm in women; serum triglyceride of at least150 mg/dL; HDL-C less than 40 mg/dL in men or less than 50 mg/dL inwomen; blood pressure of at least 130/85 mmHg; and fasting glucose of atleast 110 mg/dL. These determinants can be readily measured in clinicalpractice (JAMA, 2001, 285: 2486-2497).

“Mismatch” or “non-complementary nucleobase” refers to the case when anucleobase of a first nucleic acid is not capable of pairing with thecorresponding nucleobase of a second or target nucleic acid.

“Mixed dyslipidemia” means a condition characterized by elevatedcholesterol and elevated triglycerides.

“Modified internucleoside linkage” refers to a substitution or anychange from a naturally occurring internucleoside bond. For example, aphosphorothioate linkage is a modified internucleoside linkage.

“Modified nucleobase” refers to any nucleobase other than adenine,cytosine, guanine, thymidine, or uracil. For example, 5-methylcytosineis a modified nucleobase. An “unmodified nucleobase” means the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C), and uracil (U).

“Modified nucleoside” means a nucleoside having at least one modifiedsugar moiety, and/or modified nucleobase.

“Modified nucleotide” means a nucleotide having at least one modifiedsugar moiety, modified internucleoside linkage and/or modifiednucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at leastone modified nucleotide.

“Modified sugar” refers to a substitution or change from a naturalsugar. For example, a 2′-O-methoxyethyl modified sugar is a modifiedsugar.

“Motif” means the pattern of chemically distinct regions in an antisensecompound.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage.

“Natural sugar moiety” means a sugar found in DNA (2′-H) or RNA (2′-OH).

“Nucleic acid” refers to molecules composed of monomeric nucleotides. Anucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids(DNA), single-stranded nucleic acids (ssDNA), double-stranded nucleicacids (dsDNA), small interfering ribonucleic acids (siRNA), andmicroRNAs (miRNA). A nucleic acid may also comprise a combination ofthese elements in a single molecule.

“Nucleobase” means a heterocyclic moiety capable of pairing with a baseof another nucleic acid.

“Nucleobase complementarity” refers to a nucleobase that is capable ofbase pairing with another nucleobase. For example, in DNA, adenine (A)is complementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase refers to a nucleobase of an antisense compound that iscapable of base pairing with a nucleobase of its target nucleic acid.For example, if a nucleobase at a certain position of an antisensecompound is capable of hydrogen bonding with a nucleobase at a certainposition of a target nucleic acid, then the oligonucleotide and thetarget nucleic acid are considered to be complementary at thatnucleobase pair.

“Nucleobase sequence” means the order of contiguous nucleobasesindependent of any sugar, linkage, or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugaror the sugar and the base, and not necessarily the linkage at one ormore positions of an oligomeric compound; for example nucleosidemimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl,bicyclo or tricyclo sugar mimetics such as non-furanose sugar units.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of the nucleoside.

“Nucleotide mimetic” includes those structures used to replace thenucleoside and the linkage at one or more positions of an oligomericcompound such as for example peptide nucleic acids or morpholinos(morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiesterlinkage).

“Oligomeric compound” or “oligomer” means a polymer of linked monomericsubunits which is capable of hybridizing to a region of a nucleic acidmolecule. In certain embodiments, oligomeric compounds areoligonucleosides. In certain embodiments, oligomeric compounds areoligonucleotides. In certain embodiments, oligomeric compounds areantisense compounds. In certain embodiments, oligomeric compounds areantisense oligonucleotides. In certain embodiments, oligomeric compoundsare chimeric oligonucleotides.

“Oligonucleotide” means a polymer of linked nucleosides each of whichcan be modified or unmodified, independent from one another.

“Parenteral administration” means administration through injection orinfusion. Parenteral administration includes subcutaneousadministration, intravenous administration, intramuscularadministration, intraarterial administration, intraperitonealadministration, or intracranial administration, e.g. intrathecal orintracerebroventricular administration. Administration can becontinuous, chronic, short or intermittent.

“Peptide” means a molecule formed by linking at least two amino acids byamide bonds. Peptide refers to polypeptides and proteins.

“Pharmaceutical agent” means a substance that provides a therapeuticbenefit when administered to an individual. For example, in certainembodiments, an antisense oligonucleotide targeted to ApoCIII ispharmaceutical agent.

“Pharmaceutical composition” or “composition” means a mixture ofsubstances suitable for administering to an individual. For example, apharmaceutical composition may comprise one or more active agents and apharmaceutical carrier, such as a sterile aqueous solution.

“Pharmaceutically acceptable carrier” means a medium or diluent thatdoes not interfere with the structure of the compound. Certain of suchcarriers enable pharmaceutical compositions to be formulated as, forexample, tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspension and lozenges for the oral ingestion by a subject.Certain of such carriers enable pharmaceutical compositions to beformulated for injection, infusion or topical administration. Forexample, a pharmaceutically acceptable carrier can be a sterile aqueoussolution.

“Pharmaceutically acceptable derivative” or “salts” encompassesderivatives of the compounds described herein such as solvates,hydrates, esters, prodrugs, polymorphs, isomers, isotopically labelledvariants, pharmaceutically acceptable salts and other derivatives knownin the art.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of antisense compounds, i.e., saltsthat retain the desired biological activity of the parent compound anddo not impart undesired toxicological effects thereto. The term“pharmaceutically acceptable salt” or “salt” includes a salt preparedfrom pharmaceutically acceptable non-toxic acids or bases, includinginorganic or organic acids and bases. “Pharmaceutically acceptablesalts” of the compounds described herein may be prepared by methodswell-known in the art. For a review of pharmaceutically acceptablesalts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts:Properties, Selection and Use (Wiley-VCH, Weinheim, Germany, 2002).Sodium salts of antisense oligonucleotides are useful and are wellaccepted for therapeutic administration to humans. Accordingly, in oneembodiment the compounds described herein are in the form of a sodiumsalt.

“Phosphorothioate linkage” means a linkage between nucleosides where thephosphodiester bond is modified by replacing one of the non-bridgingoxygen atoms with a sulfur atom. A phosphorothioate linkage is amodified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e. linked) nucleobasesof a nucleic acid. In certain embodiments, a portion is a defined numberof contiguous nucleobases of a target nucleic acid. In certainembodiments, a portion is a defined number of contiguous nucleobases ofan antisense compound.

“Prevent” refers to delaying or forestalling the onset or development ofa disease, disorder, or condition for a period of time from minutes toindefinitely. Prevent also means reducing risk of developing a disease,disorder, or condition.

“Prodrug” means a therapeutic agent that is prepared in an inactive formthat is converted to an active form (i.e., a drug) within the body orcells thereof by the action of endogenous enzymes or other chemicals orconditions.

“Raise” means to increase in amount. For example, to raise plasma HDLlevels means to increase the amount of HDL in the plasma.

“Ratio of TG to HDL” means the TG levels relative to HDL levels. Theoccurrence of high TG and/or low HDL has been linked to cardiovasculardisease incidence, outcomes and mortality. “Improving the ratio of TG toHDL” means to decrease TG and/or raise HDL levels.

“Reduce” means to bring down to a smaller extent, size, amount, ornumber. For example, to reduce plasma triglyceride levels means to bringdown the amount of triglyceride in the plasma.

“Region” or “target region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. For example, a target region may encompass a 3′ UTR, a5′ UTR, an exon, an intron, an exon/intron junction, a coding region, atranslation initiation region, translation termination region, or otherdefined nucleic acid region. The structurally defined regions forApoCIII can be obtained by accession number from sequence databases suchas NCBI and such information is incorporated herein by reference. Incertain embodiments, a target region may encompass the sequence from a5′ target site of one target segment within the target region to a 3′target site of another target segment within the target region.

“Ribonucleotide” means a nucleotide having a hydroxy at the 2′ positionof the sugar portion of the nucleotide. Ribonucleotides can be modifiedwith any of a variety of substituents.

“Second agent” or “second therapeutic agent” means an agent that can beused in combination with a “first agent”. A second therapeutic agent caninclude, but is not limited to, an siRNA or antisense oligonucleotideincluding antisense oligonucleotides targeting ApoCIII. A second agentcan also include anti-ApoCIII antibodies, ApoCIII peptide inhibitors,cholesterol lowering agents, lipid lowering agents, glucose loweringagents and anti-inflammatory agents.

“Segments” are defined as smaller, sub-portions of regions within anucleic acid. For example, a “target segment” means the sequence ofnucleotides of a target nucleic acid to which one or more antisensecompounds is targeted. “5′ target site” refers to the 5′-most nucleotideof a target segment. “3′ target site” refers to the 3′-most nucleotideof a target segment.

“Shortened” or “truncated” versions of antisense oligonucleotides ortarget nucleic acids taught herein have one, two or more nucleosidesdeleted.

“Side effects” means physiological responses attributable to a treatmentother than the desired effects. In certain embodiments, side effectsinclude injection site reactions, liver function test abnormalities,renal function abnormalities, liver toxicity, renal toxicity, centralnervous system abnormalities, myopathies, and malaise. For example,increased aminotransferase levels in serum may indicate liver toxicityor liver function abnormality. For example, increased bilirubin mayindicate liver toxicity or liver function abnormality.

“Single-stranded oligonucleotide” means an oligonucleotide which is nothybridized to a complementary strand.

“Specifically hybridizable” refers to an antisense compound having asufficient degree of complementarity to a target nucleic acid to inducea desired effect, while exhibiting minimal or no effects on non-targetnucleic acids under conditions in which specific binding is desired,i.e. under physiological conditions in the case of in vivo assays andtherapeutic treatments.

“Statin” means an agent that inhibits the activity of HMG-CoA reductase.

“Subcutaneous administration” means administration just below the skin.

“Subject” means a human or non-human animal selected for treatment ortherapy.

“Symptom of cardiovascular disease or disorder” means a phenomenon thatarises from and accompanies the cardiovascular disease or disorder andserves as an indication of it. For example, angina; chest pain;shortness of breath; palpitations; weakness; dizziness; nausea;sweating; tachycardia; bradycardia; arrhythmia; atrial fibrillation;swelling in the lower extremities; cyanosis; fatigue; fainting; numbnessof the face; numbness of the limbs; claudication or cramping of muscles;bloating of the abdomen; or fever are symptoms of cardiovascular diseaseor disorder.

“Targeting” or “targeted” means the process of design and selection ofan antisense compound that will specifically hybridize to a targetnucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” and “target RNA transcript” allrefer to a nucleic acid capable of being targeted by antisensecompounds.

“Therapeutic lifestyle change” means dietary and lifestyle changesintended to lower fat/adipose tissue mass and/or cholesterol. Suchchange can reduce the risk of developing heart disease, and may includesrecommendations for dietary intake of total daily calories, total fat,saturated fat, polyunsaturated fat, monounsaturated fat, carbohydrate,protein, cholesterol, insoluble fiber, as well as recommendations forphysical activity.

“Treat” refers to administering a compound of the invention to effect analteration or improvement of a disease, disorder, or condition.

“Triglyceride” or “TG” means a lipid or neutral fat consisting ofglycerol combined with three fatty acid molecules.

“Type 2 diabetes,” (also known as “type 2 diabetes mellitus”, “diabetesmellitus, type 2”, “non-insulin-dependent diabetes (NIDDM)”, “obesityrelated diabetes”, or “adult-onset diabetes”) is a metabolic disorderthat is primarily characterized by insulin resistance, relative insulindeficiency, and hyperglycemia.

“Unmodified nucleotide” means a nucleotide composed of naturallyoccurring nucleobases, sugar moieties, and internucleoside linkages. Incertain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e.β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

“Wing segment” means one or a plurality of nucleosides modified toimpart to an oligonucleotide properties such as enhanced inhibitoryactivity, increased binding affinity for a target nucleic acid, orresistance to degradation by in vivo nucleases.

Certain Embodiments

Certain embodiments provide a method of reducing ApoCIII levels in ananimal by administering a compound to the animal targeting ApoCIII,thereby decreasing ApoCIII levels. In certain embodiments, ApoCIIIlevels are reduced in the liver or small intestine.

Certain embodiments provide a method of increasing HDL levels and/orimproving the ratio of TG to HDL in an animal by administering acompound to the animal wherein the compound targets ApoCIII andincreases HDL levels and/or improves the ratio of TG to HDL.

Certain embodiments provide a method of preventing, delaying orameliorating a cardiovascular disease, disorder, condition, or symptomthereof, in an animal comprising administering a compound targetingApoCIII to the animal, wherein the compound administered to the animalprevents, treats or ameliorates the cardiovascular disease, disorder,condition or symptom in the animal by increasing HDL levels in theanimal and/or improving the ratio of TG to HDL.

Certain embodiments provide a method of preventing, delaying orameliorating a pancreatitis in an animal comprising administering acompound targeting ApoCIII to the animal, wherein the compoundadministered to the animal prevents, treats or ameliorates thepancreatitis in the animal by increasing HDL levels in the animal and/orimproving the ratio of TG to HDL. In certain embodiments, thepancreatitis is acute pancreatitis.

Certain embodiments provide a method of preventing, treating,ameliorating, delaying the onset, or reducing the risk of, acardiovascular disease, disorder or condition in an animal, comprisingof administering to the animal a compound that targets ApoCIII, whereinthe compound prevents, treats, ameliorates, delays the onset, or reducesof the risk of the cardiovascular disease, disorder or condition in theanimal by increasing HDL levels in the animal and/or improving the ratioof TG to HDL.

Certain embodiments provide a method of decreasing CETP levels byadministering a compound targeting ApoCIII to an animal.

Certain embodiments provide a method of increasing ApoA1, PON1, fatclearance, chylomicron triglyceride clearance and/or HDL byadministering a compound targeting ApoCIII to an animal. Certainembodiments provide a method for improving the ratio of TG to HDL.

In certain embodiments, the ApoCIII nucleic acid is any of the sequencesset forth in GENBANK Accession No. NM_(—)000040.1 (incorporated hereinas SEQ ID NO: 1), and GENBANK Accession No. NT_(—)033899.8 truncatedfrom nucleotides 20262640 to 20266603 (incorporated herein as SEQ ID NO:2).

In certain embodiments, the compound targeting ApoCIII is a modifiedoligonucleotide. In further embodiments, the modified oligonucleotidehas a nucleobase sequence comprising at least 8 contiguous nucleobasesof ISIS 304801 (SEQ ID NO: 3). In certain embodiments, the modifiedoligonucleotide is at least 80%, at least 85%, at least 90%, at least95%, at least 98% or at least 100% complementary to SEQ ID NO: 1 or SEQID NO: 2.

In certain embodiments, the modified oligonucleotide consists of asingle-stranded modified oligonucleotide.

In certain embodiments, the modified oligonucleotide consists of 12-30linked nucleosides.

In certain embodiments, the modified oligonucleotide consists of 20linked nucleosides.

In certain embodiments, the compound comprises at least one modifiedinternucleoside linkage. In certain embodiments, the internucleosidelinkage is a phosphorothioate internucleoside linkage.

In certain embodiments, the compound comprises at least one nucleosidecomprising a modified sugar. In certain embodiments, the at least onemodified sugar is a bicyclic sugar. In certain embodiments, the at leastone modified sugar comprises a 2′-O-methoxyethyl.

In certain embodiments, the compound comprises at least one nucleosidecomprising a modified nucleobase. In certain embodiments, the modifiednucleobase is a 5-methylcytosine.

In certain embodiments, the compound comprising modified oligonucleotidecomprises: (i) a gap segment consisting of linked deoxynucleosides; (ii)a 5′ wing segment consisting of linked nucleosides; (iii) a 3′ wingsegment consisting of linked nucleosides, wherein the gap segment ispositioned immediately adjacent to and between the 5′ wing segment andthe 3′ wing segment and wherein each nucleoside of each wing segmentcomprises a modified sugar.

In certain embodiments, the compound comprising modified oligonucleotidecomprises: (i) a gap segment consisting of 8-12 linked deoxynucleosides;(ii) a 5′ wing segment consisting of 1-5 linked nucleosides; (iii) a 3′wing segment consisting of 1-5 linked nucleosides, wherein the gapsegment is positioned immediately adjacent to and between the 5′ wingsegment and the 3′ wing segment, wherein each nucleoside of each wingsegment comprises a 2′-O-methoxyethyl sugar, wherein each cytosine is a5′-methylcytosine, and wherein each internucleoside linkage is aphosphorothioate linkage.

In certain embodiments, the compound comprising modified oligonucleotidecomprises: (i) a gap segment consisting of ten linked deoxynucleosides;(ii) a 5′ wing segment consisting of five linked nucleosides; (iii) a 3′wing segment consisting of five linked nucleosides, wherein the gapsegment is positioned immediately adjacent to and between the 5′ wingsegment and the 3′ wing segment, wherein each nucleoside of each wingsegment comprises a 2′-O-methoxyethyl sugar, wherein each cytosine is a5′-methylcytosine, and wherein each internucleoside linkage is aphosphorothioate linkage.

In certain embodiments, the modified oligonucleotide has a nucleobasesequence comprising at least 8 contiguous nucleobases of a nucleobasesequence of ISIS 304801 (SEQ ID NO: 3).

Certain embodiments provide a method of reducing the risk of acardiovascular disease in an animal by administering to the animal atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides, wherein themodified oligonucleotide is complementary to an ApoCIII nucleic acid andwherein the modified oligonucleotide increases HDL levels and/orimproves the ratio of TG to HDL. In certain embodiments, the ApoCIIInucleic acid is either SEQ ID NO: 1 or SEQ ID NO: 2. In certainembodiments, the modified oligonucleotide is at least 80%, at least 85%,at least 90%, at least 95%, at least 98% or at least 100% complementaryto SEQ ID NO: 1 or SEQ ID NO: 2. In further embodiments the modifiednucleotide comprises at least 8 contiguous nucleobases of the nucleobasesequence of ISIS 304801 (SEQ ID NO: 3).

Certain embodiments provide a method of preventing, treating,ameliorating, or reducing at least one symptom of a cardiovasculardisease in an animal, comprising administering to the animal atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and iscomplementary to an ApoCIII nucleic acid. In certain embodiments, theApoCIII nucleic acid is either SEQ ID NO: 1 or SEQ ID NO: 2. In certainembodiments, the modified oligonucleotide is at least 80%, at least 85%,at least 90%, at least 95%, at least 98% or at least 100% complementaryto SEQ ID NO: 1 or SEQ ID NO: 2. In further embodiments, the compoundadministered to the animal prevents, treats, ameliorates or reduces atleast one symptom of the cardiovascular disease by increasing HDL levelsand/or improving the ratio of TG to HDL. In further embodiments, themodified oligonucleotide comprises at least 8 contiguous nucleobases ofISIS 304801 (SEQ ID NO: 3).

In further embodiments, symptoms of a cardiovascular disease include,but are not limited to, angina; chest pain; shortness of breath;palpitations; weakness; dizziness; nausea; sweating; tachycardia;bradycardia; arrhythmia; atrial fibrillation; swelling in the lowerextremities; cyanosis; fatigue; fainting; numbness of the face; numbnessof the limbs; claudication or cramping of muscles; bloating of theabdomen; or fever.

Certain embodiments provide a method of raising HDL levels and/orimproving the ratio of TG to HDL in an animal by administering to theanimal a compound consisting of the nucleobase sequence of ISIS 304801(SEQ ID NO: 3). Further embodiments provide a method of preventing,treating, ameliorating or reducing at least one symptom of acardiovascular disease in an animal by administering to the animal acompound consisting of the nucleobase sequence of ISIS 304801 (SEQ IDNO: 3), thereby increasing the HDL levels and/or improving the ratio ofTG to HDL in the animal.

Certain embodiments provide a method of raising HDL levels and/orimproving the ratio of TG to HDL in an animal by administering to theanimal a modified oligonucleotide having the sequence of ISIS 304801,wherein the modified oligonucleotide comprises: (i) a gap segmentconsisting of ten linked deoxynucleosides; (ii) a 5′ wing segmentconsisting of five linked nucleosides; (iii) a 3′ wing segmentconsisting of five linked nucleosides, wherein the gap segment ispositioned immediately adjacent to and between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar, wherein each cytosine is a5′-methylcytosine, and wherein each internucleoside linkage is aphosphorothioate linkage.

Certain embodiments provide a method of preventing, treating,ameliorating, or reducing at least one symptom of a cardiovasculardisease in an animal by administering to the animal a modifiedoligonucleotide having the sequence of ISIS 304801 (SEQ ID NO: 3),wherein the modified oligonucleotide of the compound comprises: (i) agap segment consisting of ten linked deoxynucleosides; (ii) a 5′ wingsegment consisting of five linked nucleosides; (iii) a 3′ wing segmentconsisting of five linked nucleosides, wherein the gap segment ispositioned immediately adjacent to and between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar, wherein each cytosine is a5′-methylcytosine, and wherein each internucleoside linkage is aphosphorothioate linkage.

Certain embodiments provide a method of raising the HDL levels and/orimproving the ratio of TG to HDL in an animal by administering to theanimal a compound comprising a modified oligonucleotide consisting of 12to 30 linked nucleosides, wherein the modified oligonucleotide iscomplementary to an ApoCIII nucleic acid. In certain embodiments, theApoCIII nucleic acid is either SEQ ID NO: 1 or SEQ ID NO: 2. In certainembodiments, the modified oligonucleotide is at least 80%, at least 85%,at least 90%, at least 95%, at least 98% or at least 100% complementaryto SEQ ID NO: 1 or SEQ ID NO: 2.

Certain embodiments provide a method of preventing, treating,ameliorating or reducing at least one symptom of a cardiovasculardisease in an animal by administering to the animal a compoundcomprising a modified oligonucleotide consisting of 12 to 30 linkednucleosides, wherein the modified oligonucleotide is complementary to anApoCIII nucleic acid, and raises the HDL levels in the animal. Incertain embodiments, the ApoCIII nucleic acid is either SEQ ID NO: 1 orSEQ ID NO: 2. In certain embodiments, the modified oligonucleotide is atleast 80%, at least 85%, at least 90%, at least 95%, at least 98% or atleast 100% complementary to SEQ ID NO: 1 or SEQ ID NO: 2.

Certain embodiments provide a method of decreasing CETP levels byadministering a compound targeting ApoCIII to an animal. In certainembodiments, the compound comprises a modified oligonucleotideconsisting of 12 to 30 linked nucleosides, wherein the modifiedoligonucleotide is complementary to an ApoCIII nucleic acid. In certainembodiments, the ApoCIII nucleic acid is either SEQ ID NO: 1 or SEQ IDNO: 2. In certain embodiments, the modified oligonucleotide is at least80%, at least 85%, at least 90%, at least 95%, at least 98% or at least100% complementary to SEQ ID NO: 1 or SEQ ID NO: 2. In certainembodiments, the compound comprises a modified oligonucleotideconsisting of 12 to 30 linked nucleosides and has a nucleobase sequencecomprising at least 8 contiguous nucleobases of ISIS 304801 (SEQ ID NO:3). In certain embodiments, the compound consists of the nucleobases ofISIS 304801 (SEQ ID NO: 3).

Certain embodiments provide a method of increasing ApoA1, PON1, fatclearance, chylomicron triglyceride clearance and/or HDL byadministering a compound targeting ApoCIII to an animal. In certainembodiments, the compound comprises a modified oligonucleotideconsisting of 12 to 30 linked nucleosides, wherein the modifiedoligonucleotide is complementary to an ApoCIII nucleic acid. In certainembodiments, the ApoCIII nucleic acid is either SEQ ID NO: 1 or SEQ IDNO: 2. In certain embodiments, the modified oligonucleotide is at least80%, at least 85%, at least 90%, at least 95%, at least 98% or at least100% complementary to SEQ ID NO: 1 or SEQ ID NO: 2. In certainembodiments, the compound comprises a modified oligonucleotideconsisting of 12 to 30 linked nucleosides and has a nucleobase sequencecomprising at least 8 contiguous nucleobases of ISIS 304801 (SEQ ID NO:3). In certain embodiments, the compound consists of the nucleobases ofISIS 304801 (SEQ ID NO: 3).

In certain embodiments, the animal is human.

In certain embodiments, the cardiovascular disease is aneurysm, angina,arrhythmia, atherosclerosis, cerebrovascular disease, coronary heartdisease, hypertension, dyslipidemia, hyperlipidemia,hypertriglyceridemia or hypercholesterolemia. In certain embodiments,the dyslipidemia is chylomicronemia.

In certain embodiments, the animal is at risk for pancreatitis. Incertain embodiments, reducing ApoCIII levels in the liver and/or smallintestine prevents pancreatitis. In certain embodiments, raising HDLlevels and/or improving the ratio of TG to HDL prevents pancreatitis.

In certain embodiments, reducing ApoCIII levels in the liver and/orsmall intestine enhance clearance of postprandial triglyceride. Incertain embodiments, raising HDL levels and/or improving the ratio of TGto HDL enhance clearance of postprandial triglyceride. In certainembodiments, reducing ApoCIII levels in the liver and/or small intestinelowers postprandial triglyceride. In certain embodiments, raising HDLlevels and/or improving the ratio of TG to HDL lowers postprandialtriglyceride.

In certain embodiments, reducing ApoCIII levels in the liver and/orsmall intestine improves the ratio of HDL to TG.

In certain embodiments, the compound is parenterally administered. Infurther embodiments, the parenteral administration is subcutaneous.

In certain embodiments, the compound is co-administered with a secondagent. In certain embodiments, the second agent is a glucose-loweringagent. In certain embodiments, the second agent is an LDL, TG orcholesterol lowering agent.

In certain embodiments, the compound and the second agent areadministered concomitantly or sequentially.

In certain embodiments, the compound is a salt form. In furtherembodiments, the compound further comprises of a pharmaceuticallyacceptable carrier or diluent.

Certain embodiments provide use of a compound targeted to ApoCIII in thepreparation of a medicament for decreasing ApoCIII levels in an animal.Certain embodiments provide use of a compound targeted to ApoCIII fordecreasing ApoCIII levels in an animal. In certain embodiments, ApoCIIIlevels are decreased in the liver or small intestine. Certainembodiments provide use of a compound targeted to ApoCIII in thepreparation of a medicament for preventing, treating, ameliorating orreducing at least one symptom of a cardiovascular disease by increasingHDL levels and/or improving the ratio of TG to HDL. Certain embodimentsprovide use of a compound targeted to ApoCIII for preventing, treating,ameliorating or reducing at least one symptom of a cardiovasculardisease by increasing HDL levels and/or improving the ratio of TG toHDL. Certain embodiments provide a use of a compound targeted to ApoCIIIfor increasing HDL levels and/or improving the ratio of TG to HDL in ananimal. Certain embodiments provide a use of a compound targeted toApoCIII for the preparation of a medicament for increasing HDL levelsand/or improving the ratio of TG to HDL in an animal. In certainembodiments, the compound is at least 80%, at least 85%, at least 90%,at least 95%, at least 98% or at least 100% complementary to an ApoCIIInucleic acid sequence. In certain embodiments, the ApoCIII nucleic acidis either SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, thecompound is a modified oligonucleotide. In certain embodiments, themodified oligonucleotide has a nucleobase sequence comprising at least 8nucleobases of ISIS 304801 (SEQ ID NO: 3). In certain embodiments, themodified oligonucleotide has the nucleobase sequence of ISIS 304801 (SEQID NO: 3). Certain embodiments provide use of a compound targeted toApoCIII in the preparation of a medicament for treating an animal withpancreatitis or at risk for pancreatitis. Certain embodiments provideuse of a compound targeted to ApoCIII for treating an animal withpancreatitis or at risk for pancreatitis. Certain embodiments provideuse of a compound targeted to ApoCIII in the preparation of a medicamentfor reducing ApoCIII levels in the liver and/or small intestine. Certainembodiments provide use of a compound targeted to ApoCIII for reducingApoCIII levels in the liver and/or small intestine. Certain embodimentsprovide use of a compound targeted to ApoCIII in the preparation of amedicament for preventing pancreatitis. Certain embodiments provide useof a compound targeted to ApoCIII for preventing pancreatitis.

Certain embodiments provide use of a compound targeted to ApoCIII in thepreparation of a medicament for reducing ApoCIII levels in the liverand/or small intestine in an animal with hypertriglyceridemia. Certainembodiments provide use of a compound targeted to ApoCIII for reducingApoCIII levels in the liver and/or small intestine in an animal withhypertriglyceridemia. Certain embodiments provide use of a compoundtargeted to ApoCIII in the preparation of a medicament for enhancingclearance of postprandial triglyceride. Certain embodiments provide useof a compound targeted to ApoCIII for enhancing clearance ofpostprandial triglyceride. Certain embodiments provide use of a compoundtargeted to ApoCIII in the preparation of a medicament for loweringpostprandial triglyceride. Certain embodiments provide use of a compoundtargeted to ApoCIII for lowering postprandial triglyceride.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics,antisense compounds, antisense oligonucleotides, and siRNAs. Anoligomeric compound may be “antisense” to a target nucleic acid, meaningthat is capable of undergoing hybridization to a target nucleic acidthrough hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequencethat, when written in the 5′ to 3′ direction, comprises the reversecomplement of the target segment of a target nucleic acid to which it istargeted. In certain such embodiments, an antisense oligonucleotide hasa nucleobase sequence that, when written in the 5′ to 3′ direction,comprises the reverse complement of the target segment of a targetnucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to an ApoCIIInucleic acid is 12 to 30 nucleotides in length. In other words,antisense compounds are from 12 to 30 linked nucleobases. In otherembodiments, the antisense compound comprises a modified oligonucleotideconsisting of 8 to 80, 10 to 80, 12 to 50, 15 to 30, 18 to 24, 19 to 22,or 20 linked nucleobases. In certain such embodiments, the antisensecompound comprises a modified oligonucleotide consisting of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linkednucleobases in length, or a range defined by any two of the abovevalues. In some embodiments, the antisense compound is an antisenseoligonucleotide.

In certain embodiments, the antisense compound comprises a shortened ortruncated modified oligonucleotide. The shortened or truncated modifiedoligonucleotide can have one or more nucleosides deleted from the 5′ end(5′ truncation), one or more nucleosides deleted from the 3′ end (3′truncation) or one or more nucleosides deleted from the central portion.Alternatively, the deleted nucleosides may be dispersed throughout themodified oligonucleotide, for example, in an antisense compound havingone nucleoside deleted from the 5′ end and one nucleoside deleted fromthe 3′ end.

When a single additional nucleoside is present in a lengthenedoligonucleotide, the additional nucleoside may be located at the centralportion, 5′ or 3′ end of the oligonucleotide. When two or moreadditional nucleosides are present, the added nucleosides may beadjacent to each other, for example, in an oligonucleotide having twonucleosides added to the central portion, to the 5′ end (5′ addition),or alternatively to the 3′ end (3′ addition), of the oligonucleotide.Alternatively, the added nucleosides may be dispersed throughout theantisense compound, for example, in an oligonucleotide having onenucleoside added to the 5′ end and one subunit added to the 3′ end.

It is possible to increase or decrease the length of an antisensecompound, such as an antisense oligonucleotide, and/or introducemismatch bases without eliminating activity. For example, in Woolf etal. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series ofantisense oligonucleotides 13-25 nucleobases in length were tested fortheir ability to induce cleavage of a target RNA in an oocyte injectionmodel. Antisense oligonucleotides 25 nucleobases in length with 8 or 11mismatch bases near the ends of the antisense oligonucleotides were ableto direct specific cleavage of the target mRNA, albeit to a lesserextent than the antisense oligonucleotides that contained no mismatches.Similarly, target specific cleavage was achieved using 13 nucleobaseantisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001)demonstrated the ability of an oligonucleotide having 100%complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xLmRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and invivo. Furthermore, this oligonucleotide demonstrated potent anti-tumoractivity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a seriesof tandem 14 nucleobase antisense oligonucleotides, and 28 and 42nucleobase antisense oligonucleotides comprised of the sequence of twoor three of the tandem antisense oligonucleotides, respectively, fortheir ability to arrest translation of human DHFR in a rabbitreticulocyte assay. Each of the three 14 nucleobase antisenseoligonucleotides alone was able to inhibit translation, albeit at a moremodest level than the 28 or 42 nucleobase antisense oligonucleotides.

Antisense Compound Motifs

In certain embodiments, antisense compounds targeted to an ApoCIIInucleic acid have chemically modified subunits arranged in patterns, ormotifs, to confer to the antisense compounds properties such as enhancedinhibitory activity, increased binding affinity for a target nucleicacid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, increased binding affinity for the targetnucleic acid, and/or increased inhibitory activity. A second region of achimeric antisense compound may optionally serve as a substrate for thecellular endonuclease RNase H, which cleaves the RNA strand of anRNA:DNA duplex.

Antisense compounds having a gapmer motif are considered chimericantisense compounds. In a gapmer an internal region having a pluralityof nucleotides that supports RNase H cleavage is positioned betweenexternal regions having a plurality of nucleotides that are chemicallydistinct from the nucleosides of the internal region. In the case of anantisense oligonucleotide having a gapmer motif, the gap segmentgenerally serves as the substrate for endonuclease cleavage, while thewing segments comprise modified nucleosides. In certain embodiments, theregions of a gapmer are differentiated by the types of sugar moietiescomprising each distinct region. The types of sugar moieties that areused to differentiate the regions of a gapmer may in some embodimentsinclude β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modifiednucleosides (such 2′-modified nucleosides may include 2′-MOE, and2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (suchbicyclic sugar modified nucleosides may include those having a4′-(CH2)n-O-2′ bridge, where n=1 or n=2). Preferably, each distinctregion comprises uniform sugar moieties. The wing-gap-wing motif isfrequently described as “X—Y—Z”, where “X” represents the length of the5′ wing region, “Y” represents the length of the gap region, and “Z”represents the length of the 3′ wing region. As used herein, a gapmerdescribed as “X—Y—Z” has a configuration such that the gap segment ispositioned immediately adjacent to each of the 5′ wing segment and the3′ wing segment. Thus, no intervening nucleotides exist between the 5′wing segment and gap segment, or the gap segment and the 3′ wingsegment. Any of the antisense compounds described herein can have agapmer motif. In some embodiments, X and Z are the same; in otherembodiments they are different. In a preferred embodiment, Y is between8 and 15 nucleotides. X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30 or more nucleotides. Thus, gapmers include, but are notlimited to, for example 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5,2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 6-8-6, 5-8-5, 1-8-1,2-6-2, 2-13-2, 1-8-2, 2-8-3, 3-10-2, 1-18-2 or 2-18-2.

In certain embodiments, the antisense compound as a “wingmer” motif,having a wing-gap or gap-wing configuration, i.e. an X—Y or Y—Zconfiguration as described above for the gapmer configuration. Thus,wingmer configurations include, but are not limited to, for example5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13 or5-13.

In certain embodiments, antisense compounds targeted to an ApoCIIInucleic acid possess a 5-10-5 gapmer motif.

In certain embodiments, an antisense compound targeted to an ApoCIIInucleic acid has a gap-widened motif.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode ApoCIII include, without limitation,the following: GENBANK Accession No. NM_(—)000040.1 (incorporated hereinas SEQ ID NO: 1), and GENBANK Accession No. NT_(—)033899.8 truncatedfrom nucleotides 20262640 to 20266603 (incorporated herein as SEQ ID NO:2).

It is understood that the sequence set forth in each SEQ ID NO in theExamples contained herein is independent of any modification to a sugarmoiety, an internucleoside linkage, or a nucleobase. As such, antisensecompounds defined by a SEQ ID NO may comprise, independently, one ormore modifications to a sugar moiety, an internucleoside linkage, or anucleobase. Antisense compounds described by Isis Number (Isis No)indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined regionof the target nucleic acid. For example, a target region may encompass a3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region. The structurally defined regionsfor ApoCIII can be obtained by accession number from sequence databasessuch as NCBI and such information is incorporated herein by reference.In certain embodiments, a target region may encompass the sequence froma 5′ target site of one target segment within the target region to a 3′target site of another target segment within the target region.

In certain embodiments, a “target segment” is a smaller, sub-portion ofa target region within a nucleic acid. For example, a target segment canbe the sequence of nucleotides of a target nucleic acid to which one ormore antisense compounds are targeted. “5′ target site” refers to the5′-most nucleotide of a target segment. “3′ target site” refers to the3′-most nucleotide of a target segment.

A target region may contain one or more target segments. Multiple targetsegments within a target region may be overlapping. Alternatively, theymay be non-overlapping. In certain embodiments, target segments within atarget region are separated by no more than about 300 nucleotides. Incertain embodiments, target segments within a target region areseparated by a number of nucleotides that is, is about, is no more than,is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30,20, or 10 nucleotides on the target nucleic acid, or is a range definedby any two of the preceding values. In certain embodiments, targetsegments within a target region are separated by no more than, or nomore than about, 5 nucleotides on the target nucleic acid. In certainembodiments, target segments are contiguous. Contemplated are targetregions defined by a range having a starting nucleic acid that is any ofthe 5′ target sites or 3′ target sites listed, herein.

Targeting includes determination of at least one target segment to whichan antisense compound hybridizes, such that a desired effect occurs. Incertain embodiments, the desired effect is a reduction in mRNA targetnucleic acid levels. In certain embodiments, the desired effect isreduction of levels of protein encoded by the target nucleic acid or aphenotypic change associated with the target nucleic acid.

Suitable target segments may be found within a 5′ UTR, a coding region,a 3′ UTR, an intron, an exon, or an exon/intron junction. Targetsegments containing a start codon or a stop codon are also suitabletarget segments. A suitable target segment may specifically exclude acertain structurally defined region such as the start codon or stopcodon.

The determination of suitable target segments may include a comparisonof the sequence of a target nucleic acid to other sequences throughoutthe genome. For example, the BLAST algorithm may be used to identifyregions of similarity amongst different nucleic acids. This comparisoncan prevent the selection of antisense compound sequences that mayhybridize in a non-specific manner to sequences other than a selectedtarget nucleic acid (i.e., non-target or off-target sequences).

There can be variation in activity (e.g., as defined by percentreduction of target nucleic acid levels) of the antisense compoundswithin an active target region. In certain embodiments, reductions inApoCIII mRNA levels are indicative of inhibition of ApoCIII expression.Reductions in levels of an ApoCIII protein can be indicative ofinhibition of target mRNA expression. Further, phenotypic changes can beindicative of inhibition of ApoCIII expression. For example, an increasein HDL levels, decrease in LDL levels, or decrease in triglyceridelevels, are among phenotypic changes that may be assayed for inhibitionof ApoCIII expression. Other phenotypic indications, e.g., symptomsassociated with a cardiovascular disease, may also be assessed; forexample, angina; chest pain; shortness of breath; palpitations;weakness; dizziness; nausea; sweating; tachycardia; bradycardia;arrhythmia; atrial fibrillation; swelling in the lower extremities;cyanosis; fatigue; fainting; numbness of the face; numbness of thelimbs; claudication or cramping of muscles; bloating of the abdomen; orfever.

Hybridization

In some embodiments, hybridization occurs between an antisense compounddisclosed herein and an ApoCIII nucleic acid. The most common mechanismof hybridization involves hydrogen bonding (e.g., Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementarynucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditionsare sequence-dependent and are determined by the nature and compositionof the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizableto a target nucleic acid are well known in the art (Sambrooke andRussell, Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001). Incertain embodiments, the antisense compounds provided herein arespecifically hybridizable with an ApoCIII nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary toeach other when a sufficient number of nucleobases of the antisensecompound can hydrogen bond with the corresponding nucleobases of thetarget nucleic acid, such that a desired effect will occur (e.g.,antisense inhibition of a target nucleic acid, such as an ApoCIIInucleic acid).

An antisense compound may hybridize over one or more segments of anApoCIII nucleic acid such that intervening or adjacent segments are notinvolved in the hybridization event (e.g., a loop structure, mismatch orhairpin structure).

In certain embodiments, the antisense compounds provided herein, or aspecified portion thereof, are, or are at least, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% complementary to an ApoCIII nucleic acid, a target region, targetsegment, or specified portion thereof. Percent complementarity of anantisense compound with a target nucleic acid can be determined usingroutine methods.

For example, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining non-complementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) non-complementary nucleobases which are flankedby two regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden,Genome Res., 1997, 7, 649 656). Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., 1981, 2, 482-489).

In certain embodiments, the antisense compounds provided herein, orspecified portions thereof, are fully complementary (i.e. 100%complementary) to a target nucleic acid, or specified portion thereof.For example, an antisense compound may be fully complementary to anApoCIII nucleic acid, or a target region, or a target segment or targetsequence thereof. As used herein, “fully complementary” means eachnucleobase of an antisense compound is capable of precise base pairingwith the corresponding nucleobases of a target nucleic acid. Forexample, a 20 nucleobase antisense compound is fully complementary to atarget sequence that is 400 nucleobases long, so long as there is acorresponding 20 nucleobase portion of the target nucleic acid that isfully complementary to the antisense compound. Fully complementary canalso be used in reference to a specified portion of the first and/or thesecond nucleic acid. For example, a 20 nucleobase portion of a 30nucleobase antisense compound can be “fully complementary” to a targetsequence that is 400 nucleobases long. The 20 nucleobase portion of the30 nucleobase oligonucleotide is fully complementary to the targetsequence if the target sequence has a corresponding 20 nucleobaseportion wherein each nucleobase is complementary to the 20 nucleobaseportion of the antisense compound. At the same time, the entire 30nucleobase antisense compound may or may not be fully complementary tothe target sequence, depending on whether the remaining 10 nucleobasesof the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase(s) can be at the 5′ endor 3′ end of the antisense compound. Alternatively, thenon-complementary nucleobase(s) can be at an internal position of theantisense compound. When two or more non-complementary nucleobases arepresent, they can be contiguous (i.e. linked) or non-contiguous. In oneembodiment, a non-complementary nucleobase is located in the wingsegment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to, 12,13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no morethan 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas an ApoCIII nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleobases in length comprise no more than 6, no more than 5, nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas an ApoCIII nucleic acid, or specified portion thereof.

The antisense compounds provided herein also include those which arecomplementary to a portion of a target nucleic acid. As used herein,“portion” refers to a defined number of contiguous (i.e. linked)nucleobases within a region or segment of a target nucleic acid. A“portion” can also refer to a defined number of contiguous nucleobasesof an antisense compound. In certain embodiments, the antisensecompounds are complementary to at least an 8 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds arecomplementary to at least a 10 nucleobase portion of a target segment.In certain embodiments, the antisense compounds are complementary to atleast a 12 nucleobase portion of a target segment. In certainembodiments, the antisense compounds are complementary to at least a 15nucleobase portion of a target segment. Also contemplated are antisensecompounds that are complementary to at least a 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment,or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percentidentity to a particular nucleotide sequence, SEQ ID NO, or sequence ofa compound represented by a specific Isis number, or portion thereof. Asused herein, an antisense compound is identical to the sequencedisclosed herein if it has the same nucleobase pairing ability. Forexample, a RNA which contains uracil in place of thymidine in adisclosed DNA sequence would be considered identical to the DNA sequencesince both uracil and thymidine pair with adenine. Shortened andlengthened versions of the antisense compounds described herein as wellas compounds having non-identical bases relative to the antisensecompounds provided herein also are contemplated. The non-identical basesmay be adjacent to each other or dispersed throughout the antisensecompound. Percent identity of an antisense compound is calculatedaccording to the number of bases that have identical base pairingrelative to the sequence to which it is being compared.

In certain embodiments, the antisense compounds, or portions thereof,are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to one or more of the antisense compounds or SEQ ID NOs, or aportion thereof, disclosed herein.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known asbase) portion of the nucleoside is normally a heterocyclic base moiety.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.Oligonucleotides are formed through the covalent linkage of adjacentnucleosides to one another, to form a linear polymeric oligonucleotide.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide.

Modifications to antisense compounds encompass substitutions or changesto internucleoside linkages, sugar moieties, or nucleobases. Modifiedantisense compounds are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for nucleic acid target, increased stability in thepresence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides can also be employed to increase thebinding affinity of a shortened or truncated antisense oligonucleotidefor its target nucleic acid. Consequently, comparable results can oftenbe obtained with shorter antisense compounds that have such chemicallymodified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′to 5′ phosphodiester linkage. Antisense compounds having one or moremodified, i.e. non-naturally occurring, internucleoside linkages areoften selected over antisense compounds having naturally occurringinternucleoside linkages because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for target nucleicacids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages includeinternucleoside linkages that retain a phosphorus atom as well asinternucleoside linkages that do not have a phosphorus atom.Representative phosphorus containing internucleoside linkages include,but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinglinkages are well known.

In certain embodiments, antisense compounds targeted to an ApoCIIInucleic acid comprise one or more modified internucleoside linkages. Incertain embodiments, the modified internucleoside linkages arephosphorothioate linkages. In certain embodiments, each internucleosidelinkage of an antisense compound is a phosphorothioate internucleosidelinkage.

Modified Sugar Moieties

Antisense compounds of the invention can optionally contain one or morenucleosides wherein the sugar group has been modified. Such sugarmodified nucleosides may impart enhanced nuclease stability, increasedbinding affinity, or some other beneficial biological property to theantisense compounds. In certain embodiments, nucleosides comprisechemically modified ribofuranose ring moieties. Examples of chemicallymodified ribofuranose rings include without limitation, addition ofsubstitutent groups (including 5′ and 2′ substituent groups, bridging ofnon-geminal ring atoms to form bicyclic nucleic acids (BNA), replacementof the ribosyl ring oxygen atom with S, N(R), or C(R₁)(R₂) (R, R₁ and R₂are each independently H, C₁-C₁₂ alkyl or a protecting group) andcombinations thereof. Examples of chemically modified sugars include2′-F-5′-methyl substituted nucleoside (see PCT International ApplicationWO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bissubstituted nucleosides) or replacement of the ribosyl ring oxygen atomwith S with further substitution at the 2′-position (see published U.S.Patent Application US2005-0130923, published on Jun. 16, 2005) oralternatively 5′-substitution of a BNA (see PCT InternationalApplication WO 2007/134181 Published on Nov. 22, 2007 wherein LNA issubstituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include withoutlimitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S,2′-F, 2′-OCH₃, 2′-OCH₂CH₃, 2′-OCH₂CH₂F and 2′-O(CH₂)₂OCH₃ substituentgroups. The substituent at the 2′ position can also be selected fromallyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF₃, OCH₂F,O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_(m))(R_(n)), O—CH₂—C(═O)—N(R_(m))(R_(n)), andO—CH₂—C(═O)—N(R_(l))—(CH₂)₂—N(R_(m))(R_(n)), where each R_(l), R_(m) andR_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosidescomprising a bicyclic sugar moiety. Examples of bicyclic nucleosidesinclude without limitation nucleosides comprising a bridge between the4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisensecompounds provided herein include one or more bicyclic nucleosidescomprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclicnucleosides, include but are not limited to one of the formulae:4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof see U.S. Pat. No.7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogsthereof see published International Application WO/2009/006478,published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof seepublished International Application WO/2008/150729, published Dec. 11,2008); 4′-CH₂—O—N(CH₃)-2′ (see published U.S. Patent ApplicationUS2004-0171570, published Sep. 2, 2004); 4′-CH₂—N(R)—O-2′, wherein R isH, C₁-C₁₂ alkyl, or a protecting group (see U.S. Pat. No. 7,427,672,issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see Chattopadhyaya etal., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (andanalogs thereof see published International Application WO 2008/154401,published on Dec. 8, 2008).

Further reports related to bicyclic nucleosides can also be found inpublished literature (see for example: Singh et al., Chem. Commun.,1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630;Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A, 2000, 97, 5633-5638;Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh etal., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am.Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al., Curr. OpinionInvest. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8,1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S.Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499;7,034,133; 7,053,207; 7,399,845; 7,547,684; and 7,696,345; U.S. PatentPublication No. US2008-0039618; US2009-0012281; U.S. Patent Ser. Nos.60/989,574; 61/026,995; 61/026,998; 61/056,564; 61/086,231; 61/097,787;and 61/099,844; Published PCT International applications WO 1994/014226;WO 2004/106356; WO 2005/021570; WO 2007/134181; WO 2008/150729; WO2008/154401; and WO 2009/006478. Each of the foregoing bicyclicnucleosides can be prepared having one or more stereochemical sugarconfigurations including for example α-L-ribofuranose andβ-D-ribofuranose (see PCT international application PCT/DK98/00393,published on Mar. 25, 1999 as WO 99/14226).

In certain embodiments, bicyclic sugar moieties of BNA nucleosidesinclude, but are not limited to, compounds having at least one bridgebetween the 4′ and the 2′ position of the pentofuranosyl sugar moietywherein such bridges independently comprises 1 or from 2 to 4 linkedgroups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═O)—, —C(═NR_(a))—, —C(═S)—, —O—,—Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl ora protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or —C(R_(a)R_(b))—O—N(R)—. In certainembodiments, the bridge is 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′,4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′-wherein each R is, independently, H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's havebeen incorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include, but are notlimited to, (A) α-L-methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-methyleneoxy(4′-CH₂—O-2′) BNA, (C) ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) aminooxy(4′-CH₂—O—N(R)-2′) BNA, (E) oxyamino (4′-CH₂—N(R)—O-2′) BNA, and (F)methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA, (G) methylene-thio(4′-CH₂—S-2′) BNA, (H) methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methylcarbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, and (J) propylene carbocyclic(4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is the base moiety and R is independently H, a protectinggroup or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are provided having FormulaI:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—,—CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides are provided having FormulaII:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl,substituted acyl, substituted amide, thiol or substituted thio.

In one embodiment, each of the substituted groups is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃,OC(═X)J_(c), and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) andJ_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl andX is O or NJ_(c).

In certain embodiments, bicyclic nucleosides are provided having FormulaIII:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl orsubstituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleosides are provided having FormulaIV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl,substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl orsubstituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleosides are provided having FormulaV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

q_(a), q_(b), q_(e) and q_(f) are each, independently, hydrogen,halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl,C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j),SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k),C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k),N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl orsubstituted C₁-C₁₂ alkyl.

The synthesis and preparation of the methyleneoxy (4′-CH₂—O-2′) BNAmonomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine anduracil, along with their oligomerization, and nucleic acid recognitionproperties have been described (Koshkin et al., Tetrahedron, 1998, 54,3607-3630). BNAs and preparation thereof are also described in WO98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′-CH₂—O-2′) BNA and 2′-thio-BNAs, have alsobeen prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,2219-2222). Preparation of locked nucleoside analogs comprisingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., WO 99/14226).Furthermore, synthesis of 2′-amino-BNA, a novel comformationallyrestricted high-affinity oligonucleotide analog has been described inthe art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Inaddition, 2′-amino- and 2′-methylamino-BNA's have been prepared and thethermal stability of their duplexes with complementary RNA and DNAstrands has been previously reported.

In certain embodiments, bicyclic nucleosides are provided having FormulaVI:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl,substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j),NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j),O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) orN(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(l) and q_(k) together are ═C(q_(g))(q_(h)),wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and thealkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Freier etal., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al.,J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation ofcarbocyclic bicyclic nucleosides along with their oligomerization andbiochemical studies have also been described (Srivastava et al., J. Am.Chem. Soc., 2007, 129(26), 8362-8379).

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclicnucleoside” refers to a bicyclic nucleoside comprising a furanose ringcomprising a bridge connecting two carbon atoms of the furanose ringconnects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

As used herein, “monocylic nucleosides” refer to nucleosides comprisingmodified sugar moieties that are not bicyclic sugar moieties. In certainembodiments, the sugar moiety, or sugar moiety analogue, of a nucleosidemay be modified or substituted at any position.

As used herein, “2′-modified sugar” means a furanosyl sugar modified atthe 2′ position. In certain embodiments, such modifications includesubstituents selected from: a halide, including, but not limited tosubstituted and unsubstituted alkoxy, substituted and unsubstitutedthioalkyl, substituted and unsubstituted amino alkyl, substituted andunsubstituted alkyl, substituted and unsubstituted allyl, andsubstituted and unsubstituted alkynyl. In certain embodiments, 2′modifications are selected from substituents including, but not limitedto: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)F,O(CH₂)_(n)ONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, wheren and m are from 1 to about 10. Other 2′-substituent groups can also beselected from: C₁-C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving pharmacokinetic properties, or a group for improving thepharmacodynamic properties of an antisense compound, and othersubstituents having similar properties. In certain embodiments, modifiednucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem.,1997, 272, 11944-12000). Such 2′-MOE substitution have been described ashaving improved binding affinity compared to unmodified nucleosides andto other modified nucleosides, such as 2′-O-methyl, O-propyl, andO-aminopropyl. Oligonucleotides having the 2′-MOE substituent also havebeen shown to be antisense inhibitors of gene expression with promisingfeatures for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504;Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc.Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides,1997, 16, 917-926).

As used herein, a “modified tetrahydropyran nucleoside” or “modified THPnucleoside” means a nucleoside having a six-membered tetrahydropyran“sugar” substituted in for the pentofuranosyl residue in normalnucleosides (a sugar surrogate). Modified THP nucleosides include, butare not limited to, what is referred to in the art as hexitol nucleicacid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, Bioorg. Med. Chem., 2002, 10, 841-854), fluoro HNA (F-HNA) orthose compounds having Formula VII:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula VII:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, an internucleoside linkinggroup linking the tetrahydropyran nucleoside analog to the antisensecompound or one of T_(a) and T_(b) is an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundand the other of T_(a) and T_(b) is H, a hydroxyl protecting group, alinked conjugate group or a 5′ or 3′-terminal group;

-   -   q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆        alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆        alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl; and each of        R₁ and R₂ is selected from hydrogen, hydroxyl, halogen,        substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,        OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein X is O, S or NJ₁ and        each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇is methyl. In certain embodiments, THP nucleosides of Formula VII areprovided wherein one of R₁ and R₂ is fluoro. In certain embodiments, R₁is fluoro and R₂ is H; R₁ is methoxy and R₂ is H, and R₁ ismethoxyethoxy and R₂ is H.

As used herein, “2′-modified” or “2′-substituted” refers to a nucleosidecomprising a sugar comprising a substituent at the 2′ position otherthan H or OH. 2′-modified nucleosides, include, but are not limited to,bicyclic nucleosides wherein the bridge connecting two carbon atoms ofthe sugar ring connects the 2′ carbon and another carbon of the sugarring; and nucleosides with non-bridging 2′ substituents, such as allyl,amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.2′-modified nucleosides may further comprise other modifications, forexample at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugarcomprising a fluoro group at the 2′ position.

As used herein, “2′-OMe” or “2′-OCH₃” or “2′-O-methyl” each refers to anucleoside comprising a sugar comprising an —OCH₃ group at the 2′position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or“2′-O-methoxyethyl” each refers to a nucleoside comprising a sugarcomprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides. In certain embodiments, one or more ofthe plurality of nucleosides is modified. In certain embodiments, anoligonucleotide comprises one or more ribonucleosides (RNA) and/ordeoxyribonucleosides (DNA).

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds (see for example review article:Leumann, Bioorg. Med. Chem., 2002, 10, 841-854).

Such ring systems can undergo various additional substitutions toenhance activity.

Methods for the preparations of modified sugars are well known to thoseskilled in the art.

In nucleotides having modified sugar moieties, the nucleobase moieties(natural, modified or a combination thereof) are maintained forhybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or morenucleosides having modified sugar moieties. In certain embodiments, themodified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOEmodified nucleosides are arranged in a gapmer motif. In certainembodiments, the modified sugar moiety is a bicyclic nucleoside having a(4′-CH(CH₃)—O-2′) bridging group. In certain embodiments, the(4′-CH(CH₃)—O-2′) modified nucleosides are arranged throughout the wingsof a gapmer motif. In certain embodiments, the modified sugar moiety isa cEt. In certain embodiments, the cEt modified nucleotides are arrangedthroughout the wings of a gapmer motif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic unmodified nucleobases. Both natural and modifiednucleobases are capable of participating in hydrogen bonding. Suchnucleobase modifications may impart nuclease stability, binding affinityor some other beneficial biological property to antisense compounds.Modified nucleobases include synthetic and natural nucleobases such as,for example, 5-methylcytosine (5-me-C). Certain nucleobasesubstitutions, including 5-methylcytosine substitutions, areparticularly useful for increasing the binding affinity of an antisensecompound for a target nucleic acid. For example, 5-methylcytosinesubstitutions have been shown to increase nucleic acid duplex stabilityby 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds.,Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.276-278).

Additional modified nucleobases include 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

Heterocyclic base moieties may include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Nucleobases that are particularly useful for increasing the bindingaffinity of antisense compounds include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeted to an ApoCIIInucleic acid comprise one or more modified nucleobases. In certainembodiments, gap-widened antisense oligonucleotides targeted to anApoCIII nucleic acid comprise one or more modified nucleobases. Incertain embodiments, the modified nucleobase is 5-methylcytosine. Incertain embodiments, each cytosine is a 5-methylcytosine.

Compositions and Methods for Formulating Pharmaceutical Compositions

Antisense oligonucleotides may be admixed with pharmaceuticallyacceptable active or inert substance for the preparation ofpharmaceutical compositions or formulations. Compositions and methodsfor the formulation of pharmaceutical compositions are dependent upon anumber of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Antisense compound targeted to an ApoCIII nucleic acid can be utilizedin pharmaceutical compositions by combining the antisense compound witha suitable pharmaceutically acceptable diluent or carrier.

In certain embodiments, the “pharmaceutical carrier” or “excipient” is apharmaceutically acceptable solvent, suspending agent or any otherpharmacologically inert vehicle for delivering one or more nucleic acidsto an animal. The excipient can be liquid or solid and can be selected,with the planned manner of administration in mind, so as to provide forthe desired bulk, consistency, etc., when combined with a nucleic acidand the other components of a given pharmaceutical composition. Typicalpharmaceutical carriers include, but are not limited to, binding agents(e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and othersugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate,ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.);lubricants (e.g., magnesium stearate, talc, silica, colloidal silicondioxide, stearic acid, metallic stearates, hydrogenated vegetable oils,corn starch, polyethylene glycols, sodium benzoate, sodium acetate,etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); andwetting agents (e.g., sodium lauryl sulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipients, which donot deleteriously react with nucleic acids, suitable for parenteral ornon-parenteral administration can also be used to formulate thecompositions of the present invention. Suitable pharmaceuticallyacceptable carriers include, but are not limited to, water, saltsolutions, alcohols, polyethylene glycols, gelatin, lactose, amylose,magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose, polyvinylpyrrolidone and the like.

A pharmaceutically acceptable diluent includes phosphate-buffered saline(PBS). PBS is a diluent suitable for use in compositions to be deliveredparenterally. Accordingly, in one embodiment, employed in the methodsdescribed herein is a pharmaceutical composition comprising an antisensecompound targeted to an ApoCIII nucleic acid and a pharmaceuticallyacceptable diluent. In certain embodiments, the pharmaceuticallyacceptable diluent is PBS. In certain embodiments, the antisensecompound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters, oran oligonucleotide which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to pharmaceutically acceptable salts ofantisense compounds, prodrugs, pharmaceutically acceptable salts of suchprodrugs, and other bioequivalents. Suitable pharmaceutically acceptablesalts include, but are not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an antisense compound which are cleaved by endogenousnucleases within the body, to form the active antisense compound.

Conjugated Antisense Compounds

Antisense compounds may be covalently linked to one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the resulting antisense oligonucleotides. Typical conjugategroups include cholesterol moieties and lipid moieties. Additionalconjugate groups include carbohydrates, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizinggroups that are generally attached to one or both termini of antisensecompounds to enhance properties such as, for example, nucleasestability. Included in stabilizing groups are cap structures. Theseterminal modifications protect the antisense compound from exonucleasedegradation, and can help in delivery and/or localization within a cell.The cap can be present at the 5′-terminus (5′-cap), or at the3′-terminus (3′-cap), or can be present on both termini. Cap structuresare well known in the art and include, for example, inverted deoxyabasic caps. Further 3′ and 5′-stabilizing groups that can be used tocap one or both ends of an antisense compound to impart nucleasestability include those disclosed in WO 03/004602 published on Jan. 16,2003.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity or expressionof ApoCIII nucleic acids or proteins can be tested in vitro in a varietyof cell types. Cell types used for such analyses are available fromcommercial vendors (e.g. American Type Culture Collection, Manassas,Va.; Zen-Bio, Inc., Research Triangle Park, N.C.; Clonetics Corporation,Walkersville, Md.) and cells are cultured according to the vendor'sinstructions using commercially available reagents (e.g. Invitrogen LifeTechnologies, Carlsbad, Calif.). Illustrative cell types include, butare not limited to, HepG2 cells, Hep3B cells, Huh7 (hepatocellularcarcinoma) cells, primary hepatocytes, A549 cells, GM04281 fibroblastsand LLC-MK2 cells.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisenseoligonucleotides, which can be modified appropriately for treatment withother antisense compounds.

In general, cells are treated with antisense oligonucleotides when thecells reach approximately 60-80% confluence in culture.

One reagent commonly used to introduce antisense oligonucleotides intocultured cells includes the cationic lipid transfection reagentLIPOFECTIN® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotidesare mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, Calif.)to achieve the desired final concentration of antisense oligonucleotideand a LIPOFECTIN® concentration that typically ranges 2 to 12 ug/mL per100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes LIPOFECTAMINE 2000® (Invitrogen, Carlsbad,Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE 2000® inOPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) toachieve the desired concentration of antisense oligonucleotide and aLIPOFECTAMINE® concentration that typically ranges 2 to 12 ug/mL per 100nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes Cytofectin® (Invitrogen, Carlsbad, Calif.).Antisense oligonucleotide is mixed with Cytofectin® in OPTI-MEM® 1reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve thedesired concentration of antisense oligonucleotide and a Cytofectin®concentration that typically ranges 2 to 12 ug/mL per 100 nM antisenseoligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes Oligofectamine™ (Invitrogen Life Technologies,Carlsbad, Calif.). Antisense oligonucleotide is mixed withOligofectamine™ in Opti-MEM™-1 reduced serum medium (Invitrogen LifeTechnologies, Carlsbad, Calif.) to achieve the desired concentration ofoligonucleotide with an Oligofectamine™ to oligonucleotide ratio ofapproximately 0.2 to 0.8 μL per 100 nM.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes FuGENE 6 (Roche Diagnostics Corp., Indianapolis,Ind.). Antisense oligomeric compound was mixed with FuGENE 6 in 1 mL ofserum-free RPMI to achieve the desired concentration of oligonucleotidewith a FuGENE 6 to oligomeric compound ratio of 1 to 4 μL of FuGENE 6per 100 nM.

Another technique used to introduce antisense oligonucleotides intocultured cells includes electroporation (Sambrooke and Russell inMolecular Cloning. A Laboratory Manual. Third Edition. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. 2001).

Cells are treated with antisense oligonucleotides by routine methods.Cells are typically harvested 16-24 hours after antisenseoligonucleotide treatment, at which time RNA or protein levels of targetnucleic acids are measured by methods known in the art and describedherein (Sambrooke and Russell in Molecular Cloning. A Laboratory Manual.Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. 2001). In general, when treatments are performed in multiplereplicates, the data are presented as the average of the replicatetreatments.

The concentration of antisense oligonucleotide used varies from cellline to cell line. Methods to determine the optimal antisenseoligonucleotide concentration for a particular cell line are well knownin the art (Sambrooke and Russell in Molecular Cloning. A LaboratoryManual. Third Edition. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. 2001). Antisense oligonucleotides are typically used atconcentrations ranging from 1 nM to 300 nM when transfected withLIPOFECTAMINE2000® (Invitrogen, Carlsbad, Calif.), Lipofectin®(Invitrogen, Carlsbad, Calif.) or Cytofectin™ (Genlantis, San Diego,Calif.). Antisense oligonucleotides are used at higher concentrationsranging from 625 to 20,000 nM when transfected using electroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+mRNA.Methods of RNA isolation are well known in the art (Sambrooke andRussell in Molecular Cloning. A Laboratory Manual. Third Edition. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2001). RNA isprepared using methods well known in the art, for example, using theTRIZOL® Reagent (Invitrogen, Carlsbad, Calif.) according to themanufacturer's recommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of an ApoCIII nucleic acid can beassayed in a variety of ways known in the art (Sambrooke and Russell inMolecular Cloning. A Laboratory Manual. Third Edition. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. 2001). For example,target nucleic acid levels can be quantitated by, e.g., Northern blotanalysis, competitive polymerase chain reaction (PCR), or quantitativereal-time PCR. RNA analysis can be performed on total cellular RNA orpoly(A)+ mRNA. Methods of RNA isolation are well known in the art.Northern blot analysis is also routine in the art. Quantitativereal-time PCR can be conveniently accomplished using the commerciallyavailable ABI PRISM® 7600, 7700, or 7900 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels may be accomplished by quantitativereal-time PCR using the ABI PRISM® 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. Methods of quantitative real-time PCRare well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reversetranscriptase (RT) reaction, which produces complementary DNA (cDNA)that is then used as the substrate for the real-time PCR amplification.The RT and real-time PCR reactions are performed sequentially in thesame sample well. RT and real-time PCR reagents are obtained fromInvitrogen (Carlsbad, Calif.). RT and real-time-PCR reactions arecarried out by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR can benormalized using either the expression level of a gene whose expressionis constant, such as cyclophilin A, or by quantifying total RNA usingRIBOGREEN® (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expressionis quantified by real time PCR, by being run simultaneously with thetarget, multiplexing, or separately. Total RNA is quantified usingRIBOGREEN® RNA quantification reagent (Invitrogen, Inc. Carlsbad,Calif.). Methods of RNA quantification by RIBOGREEN® are taught inJones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374). ACYTOFLUOR® 4000 instrument (PE Applied Biosystems, Foster City, Calif.)is used to measure RIBOGREEN® fluorescence.

Probes and primers are designed to hybridize to an ApoCIII nucleic acid.Methods for designing real-time PCR probes and primers are well known inthe art, and may include the use of software such as PRIMER EXPRESS®Software (Applied Biosystems, Foster City, Calif.).

Gene target quantities obtained by RT, real-time PCR can use either theexpression level of GAPDH or Cyclophilin A, genes whose expression areconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH or Cyclophilin A expression can bequantified by RT, real-time PCR, by being run simultaneously with thetarget, multiplexing, or separately. Total RNA was quantified usingRiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene,Oreg.).

Analysis of Protein Levels

Antisense inhibition of ApoCIII nucleic acids can be assessed bymeasuring ApoCIII protein levels. Protein levels of ApoCIII can beevaluated or quantitated in a variety of ways well known in the art,such as immunoprecipitation, Western blot analysis (immunoblotting),enzyme-linked immunosorbent assay (ELISA), quantitative protein assays,protein activity assays (for example, caspase activity assays),immunohistochemistry, immunocytochemistry or fluorescence-activated cellsorting (FACS) (Sambrooke and Russell in Molecular Cloning. A LaboratoryManual. Third Edition. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. 2001). Antibodies directed to a target can be identifiedand obtained from a variety of sources, such as the MSRS catalog ofantibodies (Aerie Corporation, Birmingham, Mich.), or can be preparedvia conventional monoclonal or polyclonal antibody generation methodswell known in the art. Antibodies useful for the detection of human andmouse ApoCIII are commercially available.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, antisense oligonucleotides, are testedin animals to assess their ability to inhibit expression of ApoCIII andproduce phenotypic changes. Testing can be performed in normal animals,or in experimental disease models. For administration to animals,antisense oligonucleotides are formulated in a pharmaceuticallyacceptable diluent, such as phosphate-buffered saline. Administrationincludes parenteral routes of administration. Calculation of antisenseoligonucleotide dosage and dosing frequency depends upon factors such asroute of administration and animal body weight. Following a period oftreatment with antisense oligonucleotides, RNA is isolated from tissueand changes in ApoCIII nucleic acid expression are measured. Changes inApoCIII protein levels are also measured.

Certain Indications

In certain embodiments, provided herein are methods of treating anindividual comprising administering one or more pharmaceuticalcompositions as described herein. In certain embodiments, the individualhas a cardiovascular disease or a metabolic disorder.

In certain embodiments, the cardiovascular disease is aneurysm, angina,arrhythmia, atherosclerosis, cerebrovascular disease, coronary heartdisease, hypertension, dyslipidemia, hyperlipidemia,hypertriglyceridemia, hypercholesterolemia, stroke and the like. Incertain embodiments, the dyslipidemia is chylomicronemia.

As shown in the examples below, compounds targeted to ApoCIII asdescribed herein have been shown to modulate physiological markers orphenotypes of a cardiovascular disease. In certain of the experiments,the compounds increased HDL levels, and decreased LDL and triglyceridelevels compared to untreated animals. In certain embodiments, theincrease in HDL levels and decrease in LDL and triglyceride levels wasassociated with an inhibition of ApoCIII by the compounds.

In certain embodiments, physiological markers of a cardiovasculardisease may be quantifiable. For example, HDL levels may be measured andquantified by, for example, standard lipid tests. For such markers, incertain embodiments, the marker may be increased by about 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or arange defined by any two of these values.

Also, provided herein are methods for preventing, treating orameliorating a symptom associated with a cardiovascular disease in asubject in need thereof. In certain embodiments, provided is a methodfor reducing the rate of onset of a symptom associated with acardiovascular disease. In certain embodiments, provided is a method forreducing the severity of a symptom associated with a cardiovasculardisease. In such embodiments, the methods comprise administering to anindividual in need thereof a therapeutically effective amount of acompound targeted to an ApoCIII nucleic acid.

Cardiovascular diseases are characterized by numerous physical symptoms.Any symptom known to one of skill in the art to be associated with acardiovascular disease can be prevented, treated, ameliorated orotherwise modulated as set forth above in the methods described above.In certain embodiments, the symptom may be any of, but not limited to,angina, chest pain, shortness of breath, palpitations, weakness,dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia,atrial fibrillation, swelling in the lower extremities, cyanosis,fatigue, fainting, numbness of the face, numbness of the limbs,claudication or cramping of muscles, bloating of the abdomen or fever.

In certain embodiments, the symptom is angina. In certain embodiments,the symptom is chest pain. In certain embodiments, the symptom isshortness of breath. In certain embodiments, the symptom ispalpitations. In certain embodiments, the symptom is weakness. Incertain embodiments, the symptom is dizziness. In certain embodiments,the symptom is nausea. In certain embodiments, the symptom is sweating.In certain embodiments, the symptom is tachycardia. In certainembodiments, the symptom is bradycardia. In certain embodiments, thesymptom is arrhythmia. In certain embodiments, the symptom is atrialfibrillation. In certain embodiments, the symptom is swelling in thelower extremities. In certain embodiments, the symptom is cyanosis. Incertain embodiments, the symptom is fatigue. In certain embodiments, thesymptom is fainting. In certain embodiments, the symptom is numbness ofthe face. In certain embodiments, the symptom is numbness of the limbs.In certain embodiments, the symptom is claudication or cramping ofmuscles. In certain embodiments, the symptom is bloating of the abdomen.In certain embodiments, the symptom is impaired short-term fever.

In certain embodiments, the metabolic disorders include, but are notlimited to, hyperglycemia, prediabetes, diabetes (type I and type II),obesity, insulin resistance, metabolic syndrome and diabeticdyslipidemia.

In certain embodiments, compounds targeted to ApoCIII as describedherein modulate physiological markers or phenotypes of a metabolicdisorder. In certain embodiments, physiological markers of a metabolicdisorder may be quantifiable. For example, glucose levels or insulinresistance can be measured and quantified by standard tests known in theart. For such markers, in certain embodiments, the marker may bedecreased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of thesevalues. In another example, insulin sensitivity or HDL levels can bemeasured and quantified by standard tests known in the art. For suchmarkers, in certain embodiments, the marker may be increase by about 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95or 99%, or a range defined by any two of these values.

Also, provided herein are methods for preventing, treating orameliorating a symptom associated with a metabolic disorder in a subjectin need thereof. In certain embodiments, provided is a method forreducing the rate of onset of a symptom associated with a metabolicdisorder. In certain embodiments, provided is a method for reducing theseverity of a symptom associated with a metabolic disorder. In suchembodiments, the methods comprise administering to an individual in needthereof a therapeutically effective amount of a compound targeted to anApoCIII nucleic acid.

Metabolic disorders are characterized by numerous physical symptoms. Anysymptom known to one of skill in the art to be associated with ametabolic disorder can be prevented, treated, ameliorated or otherwisemodulated as set forth above in the methods described above. In certainembodiments, the symptom may be any of, but not limited to, excessiveurine production (polyuria), excessive thirst and increased fluid intake(polydipsia), blurred vision, unexplained weight loss and lethargy.

In certain embodiments, provided are methods of treating an individualcomprising administering a therapeutically effective amount of one ormore pharmaceutical compositions as described herein. In certainembodiments, the individual has a cardiovascular disease. In certainembodiments, administration of a therapeutically effective amount of anantisense compound targeted to an ApoCIII nucleic acid is accompanied bymonitoring of ApoCIII levels or markers of cardiovascular disease,diabetes or other disease process associated with the expression ofApoCIII, to determine an individual's response to administration of theantisense compound. An individual's response to administration of theantisense compound is used by a physician to determine the amount andduration of therapeutic intervention.

In certain embodiments, administration of an antisense compound targetedto an ApoCIII nucleic acid results in reduction of ApoCIII expression byat least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95 or 99%, or a range defined by any two of these values. Incertain embodiments, ApoCIII expression is reduced to ≦50 mg/L, ≦60mg/L, ≦70 mg/L, ≦80 mg/L, ≦90 mg/L, ≦100 mg/L, ≦110 mg/L, ≦120 mg/L,≦130 mg/L, ≦140 mg/L, ≦150 mg/L, ≦160 mg/L, ≦170 mg/L, ≦180 mg/L, ≦190mg/L or ≦200 mg/L.

In certain embodiments, administration of an antisense compound targetedto an ApoCIII nucleic acid results in increase in HDL levels by at leastabout 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95or 99%, or a range defined by any two of these values.

In certain embodiments, administration of an antisense compound targetedto an ApoCIII nucleic acid results in reduction of TG (postprandial orfasting) levels by at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, or a rangedefined by any two of these values. In certain embodiments, TG(postprandial or fasting) is reduced to ≦100 mg/dL, ≦110 mg/dL, ≦120mg/dL, ≦130 mg/dL, ≦140 mg/dL, ≦150 mg/dL, ≦160 mg/dL, ≦170 mg/dL, ≦180mg/dL, ≦190 mg/dL, ≦200 mg/dL, ≦210 mg/dL, ≦220 mg/dL, ≦230 mg/dL, ≦240mg/dL, ≦250 mg/dL, ≦260 mg/dL, ≦270 mg/dL, ≦280 mg/dL, ≦290 mg/dL, ≦300mg/dL, ≦350 mg/dL, ≦400 mg/dL, ≦450 mg/dL, ≦500 mg/dL, ≦550 mg/dL, ≦600mg/dL, ≦650 mg/dL, ≦700 mg/dL, ≦750 mg/dL, ≦800 mg/dL, ≦850 mg/dL, ≦900mg/dL, ≦950 mg/dL, ≦1000 mg/dL, ≦1100 mg/dL, ≦1200 mg/dL, ≦1300 mg/dL,≦1400 mg/dL, ≦1500 mg/dL, ≦1600 mg/dL, ≦1700 mg/dL, ≦1800 mg/dL or ≦1900mg/dL.

In certain embodiments, pharmaceutical compositions comprising anantisense compound targeted to ApoCIII are used for the preparation of amedicament for treating a patient suffering or susceptible to acardiovascular disease.

Administration

The compounds or pharmaceutical compositions of the present inventioncan be administered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be oral or parenteral.

In certain embodiments, the compounds and compositions as describedherein are administered parenterally. Parenteral administration includesintravenous, intra-arterial, subcutaneous, intraperitoneal orintramuscular injection or infusion.

In certain embodiments, parenteral administration is by infusion.Infusion can be chronic or continuous or short or intermittent. Incertain embodiments, infused pharmaceutical agents are delivered with apump. In certain embodiments, the infusion is intravenous.

In certain embodiments, parenteral administration is by injection. Theinjection can be delivered with a syringe or a pump. In certainembodiments, the injection is a bolus injection. In certain embodiments,the injection is administered directly to a tissue or organ. In certainembodiments, parenteral administration is subcutaneous.

In certain embodiments, formulations for parenteral administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

In certain embodiments, formulations for oral administration of thecompounds or compositions of the invention can include, but is notlimited to, pharmaceutical carriers, excipients, powders or granules,microparticulates, nanoparticulates, suspensions or solutions in wateror non-aqueous media, capsules, gel capsules, sachets, tablets orminitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In certain embodiments,oral formulations are those in which compounds of the invention areadministered in conjunction with one or more penetration enhancers,surfactants and chelators.

Dosing

In certain embodiments, pharmaceutical compositions are administeredaccording to a dosing regimen (e.g., dose, dose frequency, and duration)wherein the dosing regimen can be selected to achieve a desired effect.The desired effect can be, for example, reduction of ApoCIII or theprevention, reduction, amelioration or slowing the progression of adisease or condition associated with ApoCIII.

In certain embodiments, the variables of the dosing regimen are adjustedto result in a desired concentration of pharmaceutical composition in asubject. “Concentration of pharmaceutical composition” as used withregard to dose regimen can refer to the compound, oligonucleotide, oractive ingredient of the pharmaceutical composition. For example, incertain embodiments, dose and dose frequency are adjusted to provide atissue concentration or plasma concentration of a pharmaceuticalcomposition at an amount sufficient to achieve a desired effect.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Dosing is also dependent on drug potency andmetabolism. In certain embodiments, dosage is from 0.01 μg to 100 mg perkg of body weight, or within a range of 0.001 mg-1000 mg dosing, and maybe given once or more daily, weekly, monthly or yearly, or even onceevery 2 to 20 years. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 mg per kgof body weight, once or more daily, to once every 20 years or rangingfrom 0.001 mg to 1000 mg dosing.

Certain Combination Therapies

In certain embodiments, a first agent comprising the compound describedherein is co-administered with one or more secondary agents. In certainembodiments, such second agents are designed to treat the same disease,disorder, or condition as the first agent described herein. In certainembodiments, such second agents are designed to treat a differentdisease, disorder, or condition as the first agent described herein. Incertain embodiments, a first agent is designed to treat an undesiredside effect of a second agent. In certain embodiments, second agents areco-administered with the first agent to treat an undesired effect of thefirst agent. In certain embodiments, such second agents are designed totreat an undesired side effect of one or more pharmaceuticalcompositions as described herein. In certain embodiments, second agentsare co-administered with the first agent to produce a combinationaleffect. In certain embodiments, second agents are co-administered withthe first agent to produce a synergistic effect. In certain embodiments,the co-administration of the first and second agents permits use oflower dosages than would be required to achieve a therapeutic orprophylactic effect if the agents were administered as independenttherapy.

In certain embodiments, one or more compositions described herein andone or more other pharmaceutical agents are administered at the sametime. In certain embodiments, one or more compositions of the inventionand one or more other pharmaceutical agents are administered atdifferent times. In certain embodiments, one or more compositionsdescribed herein and one or more other pharmaceutical agents areprepared together in a single formulation. In certain embodiments, oneor more compositions described herein and one or more otherpharmaceutical agents are prepared separately.

In certain embodiments, second agents include, but are not limited to,ApoCIII lowering agent, cholesterol lowering agent, non-HDL lipidlowering (e.g., LDL) agent, HDL raising agent, fish oil, niacin,fibrate, statin, DCCR (salt of diazoxide), glucose-lowering agent and/oranti-diabetic agents. In certain embodiments, the first agent isadministered in combination with the maximally tolerated dose of thesecond agent. In certain embodiments, the first agent is administered toa subject that fails to respond to a maximally tolerated dose of thesecond agent.

Examples of ApoCIII lowering agents include an ApoCIII antisenseoligonucleotide different from the first agent, niacin or an Apo Bantisense oligonucleotide.

Examples of glucose-lowering and/or anti-diabetic agents include, but isnot limited to, a therapeutic lifestyle change, PPAR agonist, adipeptidyl peptidase (IV) inhibitor, a GLP-1 analog, insulin or aninsulin analog, an insulin secretagogue, a SGLT2 inhibitor, a humanamylin analog, a biguanide, an alpha-glucosidase inhibitor, metformin,sulfonylurea, rosiglitazone, meglitinide, thiazolidinedione,alpha-glucosidase inhibitor and the like. The sulfonylurea can beacetohexamide, chlorpropamide, tolbutamide, tolazamide, glimepiride, aglipizide, a glyburide, or a gliclazide. The meglitinide can benateglinide or repaglinide. The thiazolidinedione can be pioglitazone orrosiglitazone. The alpha-glucosidase can be acarbose or miglitol.

The cholesterol or lipid lowering therapy can include, but is notlimited to, a therapeutic lifestyle change, statins, bile acidssequestrants, nicotinic acid and fibrates. The statins can beatorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin andsimvastatin and the like. The bile acid sequestrants can be colesevelam,cholestyramine, colestipol and the like. The fibrates can begemfibrozil, fenofibrate, clofibrate and the like.

HDL increasing agents include cholesteryl ester transfer protein (CETP)inhibiting drugs (such as Torcetrapib), peroxisome proliferationactivated receptor agonists, Apo-A1, Pioglitazone and the like.

Certain Treatment Populations

Certain subjects with high TG levels are at a significant risk ofcardiovascular and metabolic disease. In many subjects with high TG(e.g., hypertriglyceridemia), current treatments cannot reduce their TGlevels to safe levels. ApoCIII plays an important role in TG metabolismand is an independent risk factor for cardiovascular disease. ApoCIIIinhibition, as shown herein, significantly decreases TG levels which canameliorate cardiovascular or metabolic disease, or the risk thereof.

Borderline high TG levels (150-199 mg/dL) are commonly found in thegeneral population and are a common component of the metabolicsyndrome/insulin resistance states. High plasma TG level of ≧200 mg/dLis a common clinical trait associated with an increased risk ofcardiovascular disease (Hegele et al., Hum Mol Genet 2009, 18:4189-4194;Hegele and Pollex, Mol Cell Biochem, 2009, 326:35-43). Very high TGlevels (≧500 and ≦2000 mg/dL) are most often associated with elevatedchylomicron levels as well, and are accompanied by increasing risk foracute pancreatitis.

In certain embodiments, the compounds, compositions and methodsdisclosed herein are used to treat subjects with a TG level between100-200 mg/dL, 100-300 mg/dL, 100-400 mg/dL, 100-500 mg/dL, 200-500mg/dL, 300-500 mg/dL, 400-500 mg/dL, 500-1000 mg/dL, 600-1000 mg/dL,700-1000 mg/dL, 800-1000 mg/dL, 900-1000 mg/dL, 500-1500 mg/dL,1000-1500 mg/dL, 100-2000 mg/dL, 150-2000 mg/dL, 200-2000 mg/dL,300-2000 mg/dL, 400-2000 mg/dL, 500-2000 mg/dL, 600-2000 mg/dL, 700-2000mg/dL, 800-2000 mg/dL, 900-2000 mg/dL, 1000-2000 mg/dL, 1100-2000 mg/dL,1200-2000 mg/dL, 1300-2000 mg/dL, 1400-2000 mg/dL, or 1500-2000 mg/dL.In certain embodiments, treatment with the compounds disclosed herein isindicated for a subject with a TG level of ≧100 mg/dL, ≧110 mg/dL, ≧120mg/dL, ≧130 mg/dL, ≧140 mg/dL, ≧150 mg/dL, ≧160 mg/dL, ≧170 mg/dL, ≧180mg/dL, ≧190 mg/dL, ≧200 mg/dL, ≧300 mg/dL, ≧400 mg/dL, ≧500 mg/dL, ≧600mg/dL, ≧700 mg/dL, ≧800 mg/dL, ≧900 mg/dL, ≧1000 mg/dL, ≧1100 mg/dL,≧1200 mg/dL, ≧1300 mg/dL, ≧1400 mg/dL, ≧1500 mg/dL, ≧1600 mg/dL, ≧1700mg/dL, ≧1800 mg/dL, ≧1900 mg/dL, ≧2000 mg/dL, ≧2100 mg/dL, ≧2200 mg/dL,≧2300 mg/dL, ≧2400 mg/dL or ≧2500 mg/dL.

Some types of hypertriglyceridemia can be characterized by theFredrickson classification system or by the classification systemdescribed by Tremblay (Tremblay et al., J Clin Lipidol, 2011, 5:37-44).In certain embodiments, the compounds, compositions and methodsdescribed herein are useful in treating subjects with, or at risk for,Fredrickson Type II, IV or V hypertriglyceridemia.

Fredrickson Type IIb (also known as familial combinedhyperlipoproteinemia) is a mixed hyperlipidemia (high cholesterol and TGlevels), caused by elevations in LDL-C and in VLDL. The high VLDL levelsare due to overproduction of substrates, including TG, acetyl CoA, andan increase in B-100 synthesis. They may also be caused by the decreasedclearance of LDL. Prevalence in the population is about 10%.

Fredrickson Type IV (also known as familial hypertriglyceridemia) is anautosomal dominant condition occurring in approximately 1% of thepopulation. TG levels are elevated as a result of excess hepaticproduction of VLDL or heterozygous LPL deficiency, but are almost alwaysless than 1000 mg/dL. Serum cholesterol levels are usually within normallimits. The disorder is heterogeneous and the phenotype stronglyinfluenced by environmental factors, particularly carbohydrate andethanol consumption. In certain embodiments, the compounds, compositionsand methods described herein are useful in treating subjects with a TGlevel ≧200 mg/dL and heterozygous LPL deficiency or VLDL overproduction.In certain embodiments, the compounds, compositions and methodsdescribed herein are useful in treating subjects with a TG level ≧500mg/dL and heterozygous LPL deficiency or VLDL overproduction.

Fredrickson Type V has high VLDL and chylomicrons. It is characterizedby carriers of loss-of-function LPL gene variants associated with LPLactivity of at least 20% (i.e. partial LPL deficiency). These subjectspresent with lactescent plasma and severe hypertriglyceridemia becauseof chylomicrons and VLDL. TG levels are invariably greater than 1000mg/dL and total cholesterol levels are always elevated. The LDL-C levelis usually low. It is also associated with increased risk for acutepancreatitis, glucose intolerance and hyperuricemia. Symptoms generallypresent in adulthood (>35 years) and, although the prevalence isrelatively rare, it is much more common than homozygous or compoundheterozygous LPL deficient subjects. In certain embodiments, thecompounds, compositions and methods described herein are useful intreating subjects with ≧1000 mg/dL TG. In certain embodiments, thecompounds, compositions and methods described herein are useful intreating subjects with, or at risk for, pancreatitis associated withhigh TG levels in a subject. In certain embodiments, the compounds,compositions and methods described herein are useful in treatingsubjects with, or at risk for, cardiovascular or metabolic diseaseassociated with high TG levels in a subject. In certain embodiments, thecardiovascular disease is aneurysm, angina, arrhythmia, atherosclerosis,cerebrovascular disease, coronary heart disease, hypertension,dyslipidemia, hyperlipidemia, hypertriglyceridemia,hypercholesterolemia, stroke and the like. In certain embodiments, thedyslipidemia is chylomicronemia. In certain embodiments, the metabolicdiseases or disorders include, but are not limited to, hyperglycemia,prediabetes, diabetes (type I and type II), obesity, insulin resistance,metabolic syndrome and diabetic dyslipidemia.

In certain embodiments, treatment with the compounds disclosed herein isindicated for a human animal with a genetic defect that increasesApoCIII levels and/or triglyceride levels. In certain embodiments, thegenetic defect is an allelic variant or polymorphism that increasesApoCIII expression. In certain embodiments, the polymorphism are T (atposition 74) to A, C (at position −641) to A, G (at position −630) to A,T (at position −625) to deletion, C (at position −482) to T, T (atposition −455) to C, C (at position 1100) to T, C (at position 3175) toG, T (at position 3206) to G, C (at position 3238) to G, and the like.In certain embodiments, the genetic defect is a heterozygous LPLdeficiency.

In certain embodiments, treatment with the compounds disclosed herein isindicated for a human animal with elevated ApoCIII levels. In certainembodiments, the elevated ApoCIII level is ≧50 mg/L, ≧60 mg/L, ≧70 mg/L,≧80 mg/L, ≧90 mg/L, ≧100 mg/L, ≧110 mg/L, ≧120 mg/L, ≧130 mg/L, ≧140mg/L, ≧150 mg/L, ≧160 mg/L, ≧170 mg/L, ≧180 mg/L, ≧190 mg/L, ≧200 mg/L,≧300 mg/L, ≧400 mg/L or ≧500 mg/L.

EXAMPLES Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the referencesrecited in the present application is incorporated herein by referencein its entirety.

Example 1 Effect of In Vivo Antisense Inhibition of Human ApoCIII inhuApoCIII Transgenic Mice

Transgenic mice with the human ApoCIII transgene utilized in the studywere the progeny of huApoCIII transgenic F1 hybrids (JacksonLaboratories, CA) and C57BL/6 mice. ISIS 304801 (previously disclosed inU.S. Pat. No. 7,598,227) with a start site of 508 on SEQ ID NO: 1(GENBANK Accession No. NM_(—)000040.1) and a start site of 3139 on SEQID NO: 2 (GENBANK Accession NT_(—)033899.8 truncated from nucleotides20263040 to 20266203), with the sequence 5′-AGCTTCTTGTCCAGCTTTAT-3′ (SEQID NO: 3) and a 5-10-5 MOE gapmer motif was utilized in this assay.Another ISIS antisense oligonucleotide, ‘Compound X’, with a 5-10-5 MOEgapmer motif, targeting another region of SEQ ID NO: 1 or SEQ ID NO: 2,was also included in this assay. Another ISIS antisense oligonucleotide,‘Compound Y’, with a 5-10-5 MOE gapmer motif, targeting a rodent ApoCIIIsequence (GenBank Accession No. NM_(—)023114.3; SEQ ID NO: 5) was alsoincluded in this assay.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/darkcycle and fed ad libitum Teklad lab chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. Antisense oligonucleotides (ASOs) were prepared in PBS andsterilized by filtering through a 0.2 micron filter. Oligonucleotideswere dissolved in 0.9% PBS for injection.

Male and female mice were assayed separately. The male mice were dividedinto three treatment groups consisting of 5 mice each. Two such groupsreceived subcutaneous injections of ISIS 304801 or Compound X at a doseof 37.5 mg/kg twice a week for 2 weeks. One group of mice receivedsubcutaneous injections of PBS twice a week for 2 weeks. The female micewere divided into four treatment groups consisting of 4-5 mice each.Three such groups received subcutaneous injections of ISIS 304801,Compounds X or Y at a dose of 37.5 mg/kg twice a week for 2 weeks. Onegroup of mice received subcutaneous injections of PBS twice a week for 2weeks. Prior to the treatment as well as after the last dose, blood waswithdrawn from each mouse and plasma samples analyzed. Two daysfollowing the final dose, the mice were euthanized, organs harvested andanalyses done.

Cholesterol and Triglyceride Levels

Plasma triglycerides and cholesterol were extracted by the method ofBligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J. Biochem. Physiol.37: 911-917, 1959) and measured with a commercially availabletriglyceride kit (DCL Triglyceride Reagent; Diagnostic Chemicals Ltd.).

The results of the triglyceride analyses in males and females arepresented in Tables 1 and 2, and are expressed in mg/dL. As observed,triglyceride levels in all the treatment groups were significantlylowered compared to that in the control groups.

For measuring the different fractions of cholesterol (HDL, LDL andVLDL), the plasma samples from the female groups were analyzed by HPLCand are presented in Table 3. As observed, antisense inhibition ofApoCIII significantly decreased VLDL and also significantly increasedlevels of HDL. An increase in HDL and a decrease in VLDL levels is acardiovascular beneficial effect of antisense inhibition of ApoCIII andcan be beneficial to animals with, or at risk or, dyslipidemic diseases.

TABLE 1 Effect of antisense oligonucleotide treatment on triglyceridelevels (mg/dL) in female transgenic mice Week 0 Week 2 % change PBS 21442533 +21 Compound X 2385 677 −72 Compound Y 2632 1644 −37 ISIS 3048012390 542 −75

TABLE 2 Effect of antisense oligonucleotide treatment on triglyceridelevels (mg/dL) in male transgenic mice Week 0 Week 2 % Change PBS 61917073 +14 ISIS 304801 6588 780 −88 Compound X 5464 861 −84

TABLE 3 Effect of antisense oligonucleotide treatment on plasmacholesterol fractions (% total cholesterol) in female transgenic miceVLDL (%) LDL (%) HDL (%) PBS 77 ± 2.6 4 ± 1.0 19 ± 1.9 ISIS 304801 41 ±0.6 7 ± 0.3 52 ± 0.5 Compound X 46 ± 5.1 7 ± 1.0 48 ± 5.8

Example 2 Dose-Dependent Antisense Inhibition of Human ApoCIII inhuApoCIII Transgenic Mice

ISIS 304801 and Compound X were further studied in a dose-dependentstudy using human ApoCIII transgenic mice.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/darkcycle and fed ad libitum Teklad lab chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. Antisense oligonucleotides (ASOs) were prepared in PBS andsterilized by filtering through a 0.2 micron filter. Oligonucleotideswere dissolved in 0.9% PBS for injection.

Female mice were divided into nine treatment groups consisting of 3 miceeach. Eight such groups received subcutaneous injections of ISIS 304801or compound X at a dose of 1.5 mg/kg/week, 5 mg/kg/week, 15 mg/kg/week,or 50 mg/kg/week for 2 weeks. One group of mice received subcutaneousinjections of PBS for 2 weeks. Prior to the treatment as well as afterthe last dose, blood was withdrawn from each mouse and plasma samplesanalyzed. Two days following the final dose, the mice were euthanized,organs harvested and analyses done.

Cholesterol and Triglyceride Levels

Plasma triglycerides and cholesterol were extracted by the method ofBligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J. Biochem. Physiol.37: 911-917, 1959) and measured with a commercially availabletriglyceride kit (DCL Triglyceride Reagent, Diagnostic Chemicals Ltd.).

The results of the cholesterol and triglyceride analyses in the mice arepresented in Tables 4 and 5, and are expressed in mg/dL. As observed,HDL levels in mice treated with higher doses of ISIS 304801 weresignificantly elevated, indicating the beneficial effect of inhibitionof ApoCIII by the oligonucleotides. LDL and triglyceride levels in thehigh dose treatment groups were lowered compared to that in the controlgroups. An increase in HDL and a decrease in LDL and TG levels is acardiovascular beneficial effect of antisense inhibition of ApoCIII andcan be beneficial to animals with, or at risk of, dyslipidemic diseases.

TABLE 4 Effect of antisense oligonucleotide treatment on cholesterol andtriglyceride levels (mg/dL) in transgenic mice Dose Total (mg/kg/wk)Cholesterol Triglycerides PBS — 124 1017 ISIS 304801 50.0 105 417 15.0116 593 5.0 101 871 1.5 125 1092 Compound X 50.0 90 496 15.0 127 11685.0 166 1506 1.5 168 1518

TABLE 5 Effect of antisense oligonucleotide treatment on HDL and LDLcholesterol levels (mg/dL) in transgenic mice Dose (mg/kg/wk) HDL LDLPBS — 40 ± 8 42 ± 8 ISIS 304801 50.0  62 ± 19 28 ± 7 15.0 60 ± 9 34 ± 75.0 44 ± 3  30 ± 13 1.5 39 ± 2 40 ± 2 Compound X 50.0  46 ± 10 25 ± 315.0 37 ± 7 40 ± 2 5.0  40 ± 10 47 ± 5 1.5 45 ± 7 44 ± 6

Example 3 Effect of Antisense Inhibition of ApoCIII in CETP TransgenicLDL Receptor Null Mice

Compound Y was further studied in a human CETP transgenic LDLr^(−/−)mouse model to examine the effects of a mouse ApoCIII antisenseinhibitor on plasma lipids and lipoprotein metabolism in hyperlipidemicmice.

Treatment

Human CETP transgenic LDLr^(−/−) transgenic mice were maintained on a12-hour light/dark cycle and fed ad libitum a western diet (42% caloriesfrom fat, 0.2% cholesterol). Animals were acclimated to this diet for 10days in the research facility before initiation of the experiment.Antisense oligonucleotides (ASOs) were prepared in PBS and sterilized byfiltering through a 0.2 micron filter. Oligonucleotides were dissolvedin 0.9% PBS for injection.

Eight week old male mice were divided into three treatment groups. Onesuch group of 6 mice received subcutaneous injections of compound Y at adose of 12.5 mg/kg/week for 4 weeks. One group of 4 mice receivedsubcutaneous injections of the control oligonucleotide ISIS 141923 (SEQID NO: 4) at a dose of 12.5 mg/kg/week for 4 weeks. One group of 5 micereceived subcutaneous injections of PBS for 4 weeks. Plasma samples weretaken at prior to the start of dosing, and at 2 and 4 weeks oftreatment.

Cholesterol and Triglyceride Levels

Plasma triglycerides and cholesterol were extracted by the method ofBligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J. Biochem. Physiol.37: 911-917, 1959) and measured with a commercially availabletriglyceride kit (DCL Triglyceride Reagent; Diagnostic Chemicals Ltd.).

The results of the cholesterol and triglyceride analyses in the mice arepresented in Tables 6 to 7, and are expressed in mg/dL. Cholesterol andtriglyceride levels in the treatment group were significantly loweredcompared to those in the control group. A decrease in cholesterol and TGlevels is a cardiovascular beneficial effect of antisense inhibition ofApoCIII and can be beneficial to animals with, or at risk of,dyslipidemic diseases.

TABLE 6 Effect of antisense oligonucleotide treatment on cholesterollevels (mg/dL) in transgenic mice Week PBS Compound Y 0 1851 1747 2 2035878 4 2359 686

TABLE 7 Effect of antisense oligonucleotide treatment on triglyceridelevels (mg/dL) in transgenic mice Week PBS Compound Y 0 297 451 2 420150 4 496 86

Inhibition of CETP Protein Levels and Activity

Plasma CETP protein levels were measured using a commercial ELISA kit(ALPCO, Cat#47-CETHU-E01). CETP protein activity was measured using afluorometric assay kit (Roar Biomedical, Inc. Cat# RB-CETP). Aspresented in Table 8, treatment with antisense oligonucleotide reducedCETP protein expression and activity. CETP (cholesterol ester transferprotein) facilitates the exchange of triglycerides and cholesterolesters between high density lipoproteins (HDL) and apoB-containinglipoproteins, such as very low density lipoproteins (VLDL), LDL andchylomicrons. A decrease in CETP is associated with increased HDL levelsand decreased LDL levels (Barter P. J. et al. Artherioscler. Thromb.Vasc. Biol. 23: 160-167, 2003). Therefore, inhibition of CETP proteinlevels and activity is a cardiovascular beneficial effect of antisenseinhibition of ApoCIII and can be beneficial to animals with, or at riskof, dyslipidemic diseases. The control oligonucleotide did not have anysignificant effect on CETP, as expected.

TABLE 8 Percent inhibition of CETP protein in transgenic mice LevelActivity Compound Y 24 24 ISIS 141923 0 3Increase of apoA1 Protein Levels and Paraoxanase-1 (PON1) Activity

Plasma ApoA1 protein levels were measured by ELISA. PON1 proteinactivity was measured using a EnzChek® Paroxanase fluorometric assay kit(Invitrogen, Cat# E33702). As presented in Tables 9 and 10, treatmentwith antisense oligonucleotide enhanced ApoA1 protein expression andincreased PON1 protein activity. ApoA1 and PON1 are major proteincomponents of HDL in plasma (Aviram, M and Rosenblat, M. Curr. Opin.Lipidol. 16: 393-399, 2005). Therefore, enhancement of protein level andactivity of these two protein components is a cardiovascular beneficialeffect of antisense inhibition of ApoCIII and can be beneficial toanimals with, or at risk of, dyslipidemic diseases. The controloligonucleotide did not have any effect on either protein, as expected.

TABLE 9 Percent increase in APOA1 protein levels in transgenic micemg/dL PBS 65 Compound Y 211 ISIS 141923 106

TABLE 10 Percent increase in PON1 protein activity in transgenic miceMinutes PBS Compound Y ISIS 141923 15 0.2 0.5 0.2 30 0.8 1.1 0.8 60 2.33.5 2.3 120 4.9 7.5 4.8 180 7.7 11.6 7.6

Example 4 Effect of Antisense Inhibition of ApoCIII on HDL CholesterolClearance in CETP Transgenic LDL Receptor Null Mice

Compound Y was further studied in a human CETP transgenic LDLr^(−/−)mouse model to examine the effects of an ApoCIII antisense inhibitor onHDL cholesterol clearance and metabolism in hyperlipidemic mice.

Treatment

Human CETP transgenic LDLr^(−/−) mice were maintained on a 12-hourlight/dark cycle and fed ad libitum a western diet (42% calories fromfat, 0.2% cholesterol). Animals were acclimated to this diet for 10 daysin the research facility before initiation of the experiment. Antisenseoligonucleotides (ASOs) were prepared in PBS and sterilized by filteringthrough a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBSfor injection.

Eight week old male mice were divided into three treatment groups. Onegroup of 6 mice received subcutaneous injections of compound Y at a doseof 15 mg/kg/week for 6 weeks. One group of 4 mice received subcutaneousinjections of the control oligonucleotide ISIS 141923 at a dose of 15mg/kg/week for 6 weeks. One group of 5 mice received subcutaneousinjections of PBS for 6 weeks.

Cholesterol and Triglyceride Levels

Plasma triglycerides and cholesterol were extracted by the method ofBligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J. Biochem. Physiol.37: 911-917, 1959) and measured with a commercially availabletriglyceride kit (DCL Triglyceride Reagent; Diagnostic Chemicals Ltd.).

The results of the cholesterol and triglyceride analyses in the mice arepresented in Table 11, and are expressed in mg/dL. Cholesterol andtriglyceride levels in the treatment group was significantly loweredcompared to that in the control group. A decrease in cholesterol and TGlevels is a cardiovascular beneficial effect of antisense inhibition ofApoCIII and can be beneficial to animals with, or at risk of,dyslipidemic diseases.

TABLE 11 Effect of antisense oligonucleotide treatment on cholesteroland triglyceide levels (mg/dL) in transgenic mice Total cholesterolTriglycerides PBS 2188 641 Compound Y 1402 170

HDL Clearance

Mice from all groups were injected via tail vein with 1×10⁶ dpm of³H-cholesteryl ether (³H-CEth)-labeled HDL. The radiolabeled cholesterylether is structurally similar to cholesterol but it will be trapped intissues that take it up. Therefore, the clearance of the radiolabeledcholesteryl ether from plasma and it's accumulation in the liver can beused to evaluate effects on reverse cholesterol transport. Plasmasamples were collected at 5 min, 1.5 hrs, 3 hrs, 6 hrs and 24 hrspost-injection and the radioactivity was counted using a liquidscintillation counter. At 24 hours, the mice were sacrificed and liverwere harvested. The liver samples were extracted in 2:1Chloroform/Methanol and the extract was blown down under nitrogen gas,solubilized in scintillation cocktail and counted using the same liquidscintillation counter.

The decrease in radiolabel, as presented in Table 12 is associated withthe clearance of HDL-Ceth from the plasma. The results indicate thattreatment with Compound Y lead to enhanced rate of HDL cholesterolclearance from the plasma. This was associated with the greateraccumulation of radiolabeled cholesteryl etherin the liver of CompoundY-treated mice, as presented in Table 13. Therefore, the data indicatesthat inhibition of ApoCIII in these transgenic mice improves reversecholesterol transport and, therefore, would have a beneficial effect onpatients with cardiovascular disease such as patients with adyslipidemic disease.

TABLE 12 Effect of antisense oligonucleotide treatment on plasma HDLcholesterol (% of count at 0 hrs) in transgenic mice 1.5 hr 3 hr 6 hr 24hr PBS 87 79 64 38 ISIS 141923 84 82 69 39 Compound Y 78 71 57 25

TABLE 13 Effect of antisense oligonucleotide treatment on hepatic uptakeof radiolabeled CEth in transgenic mice % Increase (dpm/g liver tissue)ISIS 141923 0 Compound Y 14

Example 5 Comparison of the Effect of Antisense Inhibition of HumanApoCIII in C57BL/6 Mice with an ApoCIII Knockout Mouse Model

ApoCIII knockout mice were obtained from Jackson Laboratories (stocknumber 002057) and were compared to ApoCIII antisense oligonucleotidetreated C57BL/6 mice. Compound Z, with a 5-10-5 MOE gapmer motif andtargeting a rodent ApoCIII sequence (GenBank Accession No.NM_(—)023114.3; SEQ ID NO: 5) was used in this study.

Antisense Oligonucleotide Treatment

C57BL/6 mice were maintained on a 12-hour light/dark cycle and fed ahigh fat diet (Harland Teklad lab chow #88137) for one week. Antisenseoligonucleotides (ASOs) were prepared in PBS and sterilized by filteringthrough a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBSfor injection. The mice were randomized based on total plasmacholesterol and triglyceride levels into groups of 6-8 mice each. Threegroups of C57BL/6 mice received weekly intraperitoneal injections ofCompound Z at doses of 3.1 mg/kg, 6.3 mg/kg, or 12.5 mg/kg for a periodof 6 weeks. A group of C57BL/6 mice received weekly intraperitonealinjections of PBS for a period of 6 weeks. The PBS group served as acontrol to which the oligonucleotide-treated groups and the ApoCIIIknockout mice were compared.

Two days after the final dose, the mice were sacrificed and organsharvested. Similar groups of mice fed a normal murine chow were alsotested.

Liver Triglycerides

Liver triglycerides were with an Olympus clinical analyzer (HitachiOlympus AU400e, Melville, N.Y.). The data is presented in Table 14 anddemonstrates that mice treated with an ApoCIII antisense oligonucleotidehave a different phenotype than ApoCIII knockout mice. The high doseApoCIII antisense oligonucleotide treated mice had liver triglyceridelevels similar to that of the PBS control. Liver triglyceride levels inthe ApoCIII knockout mice were significantly higher than in C57BL/6 micetreated with an ApoCIII antisense oligonucleotide or the PBS control.Therefore, antisense inhibition of ApoCIII had the beneficial effect oflowering the risk of liver steatosis compared to the ApoCIII knockoutmouse model.

TABLE 14 Liver triglyceride levels (mg/g liver tissue) Dose High-fat(mg/kg) diet fed PBS — 33 Compound Z 3.1 44 6.3 47 12.5  33 ApoCIII KO —60

Example 6 Effect of In Vivo Antisense Inhibition of ApoCIII in C57BL/6Mice

The effect of antisense inhibition of ApoCIII on plasma lipid levels andfat clearance was evaluated.

Treatment

Male C57/BL6 mice were maintained on a 12-hour light/dark cycle and fedad libitum a western diet (Harland Tekland 88137). Animals wereacclimated for at least 7 days in the research facility beforeinitiation of the experiment. Antisense oligonucleotides (ASOs) wereprepared in PBS and sterilized by filtering through a 0.2 micron filter.Oligonucleotides were dissolved in 0.9% PBS for injection.

Groups of 7-8 mice each received intraperitoneal injections of CompoundZ at a dose of 12.5 mg/kg/wk for 6 weeks. Another group of mice receivedintraperitoneal injections of control oligonucleotide ISIS 141923 at adose of 12.5 mg/kg/wk for 6 weeks. A third group of mice receivedintraperitoneal injections of PBS for 6 weeks. Two days after the finaldose, the mice were fasted for 4 hours, sacrificed and plasma andtissues were collected.

Inhibition of ApoCIII mRNA

Total RNA was extracted from the liver and small intestine and ApoCIIImRNA was quantitated by RT-PCR using an ApoCIII primer probe set andnormalized to cyclophilin. The results are presented in Table 15,expressed as percent inhibition of ApoCIII mRNA compared to the PBScontrol. ISIS 141923 did not cause any reduction in ApoCIII mRNA levels,as expected. The data demonstrated the significant inhibition of ApoCIIImRNA in the liver and small intestine by Compound Z compared to the PBScontrol.

Inhibition of intestinal ApoCIII expression could be important in theprevention of chylomicronemia (Chait et al., 1992, Adv Intern Med. 1992,37:249-73), a dyslipidemic state caused by improper clearance ofchylomicron triglyceride. Severe forms of chylomicronemia can lead topancreatitis, a life-threatening condition. By inhibiting intestinalApoCIII, inhibition of lipoprotein lipase would be reduced, andchylomicron triglyceride clearance would be enhanced, thereby preventingpancreatitis. In addition, inhibition of intestinal ApoCIII wouldenhance clearance of postprandial triglyceride, thereby lowering postprandial TG a known risk factor for coronary heart disease.

TABLE 15 Percent inhibition of ApoCIII mRNA relative to the PBS control% inhibition Liver 74 Small Intestine 13

Cholesterol and Triglyceride Levels

Plasma cholesterol were extracted by the method of Bligh and Dyer(Bligh, E. G. and Dyer, W. J. Can. J. Biochem. Physiol. 37: 911-917,1959) and measured with an Olympus clinical analyzer (Hitachi OlympusAU400e, Melville, N.Y.). HDL and non-HDL cholesterol were individuallymeasured by HPLC. Triglyceride levels were measured with the use of acommercially available triglyceride kit (DCL Triglyceride Reagent;Diagnostic Chemicals Ltd., Charlottetown, Canada). The results arepresented in Table 16 and are expressed in mg/dL. Treatment withCompound Z resulted in significant reduction of total cholesterol,non-HDL cholesterol and plasma triglyceride levels compared to the PBScontrol. A decrease in total cholesterol, non-HDL cholesterol and TGlevels is a cardiovascular beneficial effect of antisense inhibition ofApoCIII and can be beneficial to animals with, or at risk of,dyslipidemic diseases.

TABLE 16 Plasma cholesterol and triglyceride levels (mg/dL) in C56BL/6mice Dose Total HDL LDL VLDL Treatment (mg/kg/wk) cholesterolcholesterol cholesterol cholesterol Triglycerides PBS — 93 70 20 2.9 84ISIS 141923 12.5 97 78 19 2.1 82 Compound Z 12.5 95 75 17 3.1 70

Fat Clearance

Plasma samples were collected at 30 min, 1 hr, 2 hrs, 3 hrs, and 4 hrspost-injection and plasma total lipid content was measured with anOlympus clinical analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Thelipid level in the plasma, as presented in Table 17 was an inverseindicator of lipid clearance from the plasma. The results indicate thattreatment with Compound Z lead to enhanced rate of fat clearance fromthe plasma.

Therefore, the data indicates that inhibition of ApoCIII in thesetransgenic mice improves reverse cholesterol transport and, would have abeneficial effect on patients with cardiovascular disease.

TABLE 17 Effect of antisense oligonucleotide treatment on plasma lipid(mg/dL) in C57BL/6 mice Area under the 0 hr 0.5 hr 1 hr 2 hr 3 hr 4 hrcurve PBS 86 80 64 122 118 90 23631 ISIS 141923 115 142 124 236 225 15043677 Compound Z 66 55 96 156 151 101 28371

Example 7 Effect of In Vivo Antisense Inhibition of ApoCIII in C57BL/6Mice

The effect of antisense inhibition of ApoCIII on ApoCIII expressionlevels and fat clearance was evaluated.

Treatment

Male C57/BL6 mice were maintained on a 12-hour light/dark cycle and fedad libitum a western diet (Harland Tekland 88137). Animals wereacclimated for at least 7 days in the research facility beforeinitiation of the experiment. Antisense oligonucleotides (ASOs) wereprepared in PBS and sterilized by filtering through a 0.2 micron filter.Oligonucleotides were dissolved in 0.9% PBS for injection.

Groups of 5 mice each received intraperitoneal injections of an ApoCIIItargeting antisense oligonucleotide, Compound Z, at a dose of 12.5mg/kg/wk for 6 weeks. Another group of mice received intraperitonealinjections of control oligonucleotide ISIS 141923 at a dose of 12.5mg/kg/wk for 6 weeks. Two days after the final dose, the mice werefasted overnight, and a bolus of 200 μL of olive oil was administered byoral gavage. Following the bolus, plasma triglyceride levels weremeasured at regular intervals for 4 hours. The mice were sacrificed andplasma and tissues were collected.

Inhibition of ApoCIII mRNA

Total RNA was extracted from the liver and small intestine and ApoCIIImRNA was quantitated by RT-PCR using an ApoCIII primer probe set andnormalized to cyclophilin. The results are presented in Table 18,expressed as percent inhibition of ApoCIII mRNA compared to theoligonucleotide control. The data demonstrated the significantinhibition of ApoCIII mRNA in the liver and small intestine by CompoundZ compared to the oligonucleotide control.

As noted elsewhere herein, inhibition of intestinal ApoCIII expressioncould be important in the prevention of chylomicronemia (Chait et al.,1992, Adv Intern Med. 1992, 37:249-73), a dyslipidemic state caused byimproper clearance of chylomicron triglyceride. Severe forms ofchylomicronemia can lead to pancreatitis, a life-threatening condition.By inhibiting intestinal ApoCIII, inhibition of lipoprotein lipase wouldbe reduced, and chylomicron triglyceride clearance would be enhanced,thereby preventing pancreatitis. In addition, inhibition of intestinalApoCIII would enhance clearance of postprandial triglyceride, therebylowering post prandial TG a known risk factor for coronary heartdisease.

TABLE 18 Percent inhibition of ApoCIII mRNA relative to the controloligonucleotide treated C57/BL/6 mice Strain of mice % inhibitionC57BL/6 Liver 74 Small Intestine 60

Fat Clearance

Plasma samples were collected at 0 min, 30 min, 60 min, 120 min, 180min, and 240 min post-injection and plasma triglyceride concentrationswas measured with an Olympus clinical analyzer (Hitachi Olympus AU400e,Melville, N.Y.). The results indicate that treatment with Compound Zlead to enhanced rate of triglyceride clearance from the plasma.

This study can be compared to fat bolus clinical studies in whichpatients expressing high apo-CIII levels showed increased postprandialTG concentrations (Petersen K. F. et al., N Engl J Med 2010; 362:1082-1089).

TABLE 19 Effect of antisense oligonucleotide treatment on postprandialplasma TG (mg/dL) in C57BL/6 mice Strain 120 180 240 of mice 0 min 30min 60 min min min min C57BL/6 ISIS 141923 115 142 124 236 225 150Compound Z 66 55 96 156 151 101

Example 8 Effect of In Vivo Antisense Inhibition of ApoCIII in C57BL/6Mice

The effect of antisense inhibition of ApoCIII on fat clearance wasevaluated.

Treatment

Male C57/BL6 mice were maintained on a 12-hour light/dark cycle and fedad libitum a western diet (Harland Tekland 88137). Animals wereacclimated for at least 7 days in the research facility beforeinitiation of the experiment. Antisense oligonucleotides (ASOs) wereprepared in PBS and sterilized by filtering through a 0.2 micron filter.Oligonucleotides were dissolved in 0.9% PBS for injection.

Groups of 6 mice each received intraperitoneal injections of an ApoCIIItargeting antisense oligonucleotide, Compound Y or Compound Z, at a doseof 12.5 mg/kg/wk, 6.3 mg/kg/week or 3.1 mg/kg/week for 6 weeks. Anothergroup of mice received intraperitoneal injections of controloligonucleotide ISIS 141923 at a dose of 12.5 mg/kg/wk for 6 weeks.Another group of mice received intraperitoneal injections of PBS for 6weeks. Two days after the final dose, the mice were fasted overnight,and a bolus of 200 μL of olive oil was administered by oral gavage.Following the bolus, plasma triglyceride levels were measured at regularintervals for 4 hours.

Fat Clearance

Plasma samples were collected at 0 min, 30 min, 60 min, 120 min, 180min, and 240 min post-injection and plasma triglyceride concentrationswere measured with an Olympus clinical analyzer (Hitachi Olympus AU400e,Melville, N.Y.). The results indicate that treatment with Compound Y andCompound Z lead to enhanced rate of fat clearance from the plasma. N.d.indicates that the data set was not calculated.

This study can be compared to fat bolus clinical studies in whichpatients expressing high apo-CIII levels showed increased postprandialTG concentrations (Petersen K. F. et al., N Engl J Med 2010; 362:1082-1089)

TABLE 20 Effect of antisense oligonucleotide treatment on postprandialplasma TG (mg/dL) in C57BL/6 mice Dose 30 60 120 180 240 (mg/kg/wk) 0min min min min min min PBS — 79 104 118 126 113 116 ISIS 141923 12.5 75100 116 150 138 133 Compound Y 12.5 79 74 103 117 120 96 6.3 64 70 81 94120 112 3.1 91 85 106 139 164 133 Compound Z 12.5 73 65 84 118 94 76 6.370 73 89 117 120 89 3.1 86 98 143 137 152 128

Example 9 Effect of ISIS Antisense Oligonucleotides Targeting HumanApoCIII in Monkey Model of Hypertriglyceridemia

Rhesus monkeys maintained on a high fructose diet were treated with ISIS304801. Antisense oligonucleotide efficacy and tolerability, as well asthe pharmacological effect were evaluated.

Treatment

The monkeys were 2-4 years old and weighed between 2 and 5 kg. Themonkeys were assigned to six groups of five randomly assigned malerhesus monkeys each. About 60 g of diet (Certified Primate Diet #5048,PMI Nutrition International, Inc.) was provided to each monkey in Groups1-4 twice daily. An appropriate fructose supplement (i.e. approximately15% Kool Aid® mixture) was supplied in the morning for 16 weeks prior toantisense oligonucleotide dosing. To confirm sufficient triglyceridelevel elevations, blood samples for serum chemistry were collected fromall animals 1-2 weeks prior to dosing.

The groups of monkeys were injected subcutaneously with ISISoligonucleotide or PBS using a stainless steel dosing needle and syringeof appropriate size into one of 4 sites on the back of the monkeys; eachsite being used per dose in a clock-wise manner. Some of the groups weredosed three times a week for the first week (Days 1, 3, and 5) asloading doses, and subsequently twice a week for weeks 2-12, with 5mg/kg, 10 mg/kg, or 20 mg/kg of ISIS 304801. Two control groups of 5rhesus monkeys each were injected with PBS subcutaneously three times aweek for the first week (Days 1, 3, and 5), and subsequently twice aweek for weeks 2-12. The dosing chart is shown in Table 21. Monkeys ofGroups 1-4 were sacrificed on Day 86.

An additional high fat challenge was administered to monkeys of Groups 5and 6 in the form of a whipping cream milk shake. The milk shake wasstandardized to consist of 782 calories per m² of body surface with77.6% of calories from fat, 19.2% from carbohydrate, and 3.1% fromprotein. Monkeys of Groups 5 and 6 were fasted overnight and the milkshake was administered once on day 84 via a gastric tube. Blood wasdrawn just prior to (time=0 hours) and 1, 2, 3, 4, and 6 hours afteringestion of the fat load to assess the triglyceride excursion. Themonkeys were rested and remained otherwise fasting during the 6 hourspost-fat challenge. Monkeys of this group were sacrificed on Day 87.

TABLE 21 Groups of rhesus monkeys on a high fructose diet Weekly DoseNo. of Group Test Article (mg/kg/week) Sex Animals Toxicology groups 1PBS 0 Male 5 2 ISIS 304801 10 Male 5 3 ISIS 304801 20 Male 5 4 ISIS304801 40 Male 5 High Fat Challenge Test groups 5 PBS 0 Male 5 6 ISIS304801 40 Male 5

Hepatic Target Reduction RNA Analysis

Approximately 150 mg of liver was collected from Groups 1-4 for ApoCIIImRNA analysis at sacrifice. The liver was divided into 2 pieces andsoaked in two tubes containing RLT buffer with 1% beta-mercaptoethanol.The tissues were homogenized and ApoCIII expression was quantified byRT-PCR analysis. As shown in Table 22, treatment with ISIS 304801resulted in significant reduction of ApoCIII mRNA in comparison to thePBS control. The

TABLE 22 Percent Inhibition of ApoCIII mRNA in the rhesus monkey liverrelative to the PBS control Dose Groups (mg/kg/week) % inhibition 2 1068 3 20 78 4 40 83

Protein Analysis

Approximately 1.5 mL of blood was collected from all study animals inGroups 1-4 and placed in tubes containing K₂-EDTA and then centrifugedfor plasma separation. ApoCIII protein levels were quantified on aclinical analyzer using a commercially available turbidometric assay(Kamiya Biomedical Co., Seattle, Wash.). As shown in Table 23, treatmentwith ISIS 304801 resulted in significant reduction of ApoCIII proteinlevels in comparison to the PBS control. The kinetics of ApoCIII proteinlevel reduction was also analyzed and is presented in Table 24.

TABLE 23 Percent Inhibition of ApoCIII plasma protein levels in therhesus monkey relative to the PBS control Dose Groups (mg/kg/week) %inhibition 2 10 74 3 20 72 4 40 89

TABLE 24 ApoCIII plasma protein levels (mg/dL) on different days in therhesus monkey relative to the PBS control Dose Groups (mg/kg/wk) Day −7Day 16 Day 30 Day 86 1 — 4.0 5.5 3.8 5.7 2 10 4.8 3.2 0.2 1.5 3 20 4.53.8 0.9 1.6 4 40 5.2 2.9 0.0 0.6

Lipoprotein Particle Analysis

To establish the kinetics of plasma ApoCIII suppression, plasma sampleswere collected 7 days before the commencement of dosing, as well as ondays 16, 30 and 86 of the dosing period. The samples were subjected toNMR lipoprotein particle analysis (Liposcience, Raleigh, N.C.). Sincethere were no significant differences in ApoCIII lowering between thetreatment groups (Groups 2-4), the analyses is presented only of Group 2(treatment group receiving 10 mg/kg/week). The data is presented inTables 25 and 26.

Statistically significant mean changes from the baseline were observedin total plasma triglycerides (TG) and in VLDL and chylomicron TG of thetreatment group at day 30. At the same time, the control monkeysdemonstrated mean increases in the same parameters. Sustained treatmentwith ISIS 304801 in these fructose-fed monkeys led to time-dependentincreases in HDL cholesterol particle numbers by approximately 8 μmol/L(Tables 27) and did not produce elevations in LDL cholesterol in thesestudies (Table 28). There were no significant changes in LDL cholesterolparticle quantity over the 12 week treatment period, relative to the PBScontrol group.

At the time of sacrifice, livers were extracted using the Bligh and Dyerextraction method (Bligh E G and Dyer W J. Can J Biochem Physiol 1959;37: 911-917) and quantified using a Wako colorimentric TG assay.Antisense inhibition of ApoCIII did not increase hepatic TG accumulationin any of the treatment groups relative to the PBS control group (Table29).

TABLE 25 Change from baseline plasma TGmg/dL) on different days in therhesus monkey Dose (mg/kg/wk) Day −7 Day 16 Day 30 Day 86 PBS — 0 18 2327 ISIS 304801 10 0 −22 −32 −27

TABLE 26 Change from baseline VLDL and chylomicron TG (mg/dL) ondifferent days in the rhesus monkey Dose (mg/kg/wk) Day −7 Day 16 Day 30Day 86 PBS — 0 17 22 28 ISIS 304801 10 0 −22 −31 −26

TABLE 27 Change from baseline HDL cholesterol particles (μmol/L) ondifferent days in the rhesus monkey Dose (mg/kg/wk) Day −7 Day 16 Day 30Day 86 PBS — 0.3 −5.6 −3.5 −8.1 ISIS 304801 10 0.0 0.5 9.7 8.0

TABLE 28 Total LDL cholesterol particles (nmol/L) on different days inthe rhesus monkey Dose (mg/kg/wk) Day −7 Day 16 Day 30 Day 86 PBS — 982928 1005 1184 ISIS 304801 10 1007 938 781 910

TABLE 29 Hepatic triglyceride content in PBS control and ISIS 304801cohorts after 12 weeks in HTG rhesus monkeys Liver TG (ug/mg) AveragePBS 9 10 mg/kg/wk 16 20 mg/kg/wk 18 40 mg/kg/wk 6

Post-Prandial Plasma TG Clearance

At 10 weeks, the post-prandial plasma TG levels in monkeys from the 10mg/kg/week group (Group 2) were measured at 0 hr, 1 hr, 2 hr, 3 hr, and4 hr after providing a meal to the monkeys. As shown in Tables 30 and31, post-prandial plasma TG clearance was significantly increased, asshown by the 38% decrease in post-prandial TG area under the curve (AUC)in monkeys of the 10 mg/kg/week group.

Post-prandial TG clearance was also assessed in Groups 5 and 6 (the PBScontrol and 40 mg/kg/week group after a fat challenge) at 12 weeks. Thedata is presented in Table 32, and also indicates a significant decreasein post-prandial TG area under the curve (AUC) in monkeys of that groupcompared to the control.

TABLE 30 Plasma TG (mg/dL) in the rhesus monkey Dose (mg/kg/wk) 0 hr 1hr 2 hr 3 hr 4 hr PBS — 167 169 146 147 131 ISIS 10 105 87 95 102 80304801

TABLE 31 Post-prandial TG area under the curve (AUC) in the rhesusmonkey AUC PBS 610 ISIS 304801 376

TABLE 32 Post-prandial TG area under the curve (AUC) in the rhesusmonkey after fat challenge AUC PBS 613 ISIS 304801 405

Monkeys in the 10 mg/kg/wk group had lower fasting plasma TG levels thanthe PBS group at 10 weeks. Results in non-human primates demonstratethat antisense inhibition of ApoCIII represent an attractive therapeuticstrategy for reducing plasma TG and VLDL in dyslipidemic individuals,and treatment can concurrently raise HDL-C levels with no adverseeffects on LDL-C.

Example 10 ISIS 304801 Phase I Clinical Trial

In a double-blind, single and multiple ascending-dose (SAD and MAD)Phase 1 study, healthy subjects, aged 18 to 55 years, were randomlyassigned in a 3:1 ratio to receive ISIS 304801 or placebo (normalsaline).

SAD subjects were administered a single subcutaneous (SC) injection of50, 100, 200, or 400 mg (n=4/cohort) at the Study Center. The subjectsreturned to the Study Center for an outpatient visit on Days 4 and 8(±24 hour window) for blood sampling and for clinical evaluation. Thesubjects were followed until Day 15 when they were evaluated by atelephone interview.

MAD subjects were administered multiple SC injections at 50, 100, 200,and 400 mg at the Study Center. The subjects received a loading regimenof 3 doses the first week (Days 1, 3 and 5) followed by once weeklydosing for 3 weeks (Days 8, 15 and 22). The subjects were followed for 8weeks after their last dose of study drug. The subjects returned to theStudy Center for an outpatient visit on Days 29, 36 and 50 (±24 hourwindow) for safety and clinical laboratory evaluations and for bloodsampling for PK analysis. The subjects were followed until Day 78 (±7day window) when they were evaluated by a telephone interview.

The MAD subjects stayed at the Study Center from days −1 to 6 and days22 to 23, where they were provided the diet shown in Table 33. Thesubjects fasted for at least 12 hours before blood samples were takenfor evaluation at Days 5, 8, 15, 22, 23, 29, 36, 43 and 50 (±24 hourwindow).

TABLE 33 Study Center Patient Diet Serving Study Day Portion FoodDescription Day −1 525 ml. Thai noodles with Beef & mixed vegetable,[Admit] broccoli, bean sprout, green & red peppers 375 ml. Fresh fruitsalad 300 ml. Orange juice 1 Horse shoe cake 250 ml. 2% Milk Day 1 1Butter croissant 2 Eggs, Scrambled 250 ml. Sliced peaches with syrup 300ml. Orange juice 75 g. Grilled Chicken Breast [Sandwich] on Kaiser bun1.5 cup Cream of Mushroom Soup 1-pkg. Crackers Small Salad On sideCondiments 355 ml. Ginger Ale 75 g. Roast Beef 250 ml. Mashed Potatoes375 ml. Mixed Vegetables On side Gravy On side Garlic bread 300 ml.Apple juice 1 Carrot muffin 250 ml. 2% Milk Day 2 3 Pancakes On sideSyrup 1 Banana 300 ml. Orange juice  9″ Mesquite Chicken, Chicken,bacon, cheddar, tomato, red onion, & lettuce on whole wheat bread Onside Ranch dressing Cup Broccoli & cheese soup 1-pkg. Crackers 355 ml.Ginger ale 375 g. Beef Stir-fry & mixed vegetables, broccoli, carrots,celery, & onions 150 g. Steamed rice 1-pkg. Raisin 300 ml. Apple juice250 ml. Fresh fruit salad 300 ml. Cranberry juice Day 3 Med. Cheese &vegetable omelet on whole wheat bagel toasted 300 ml. Grape juice  9″Black Angus Steak, mozzarella, cheddar, sautéed onion & mushrooms onCheese bread On side Honey Bourbon Mustard, Zesty Grille Sauce CupChicken noodle soup 1-pkg. Crackers 355 ml. Ginger ale 400 g. BBQChicken Breast, Vegetables, cooked mixed, cauliflower, carrots, red &green peppers, green beans 1.5 cups Flavored rice 1-pkg. Grapes 300 ml.Apple juice 1 Raisin Oatmeal cookie 250 ml. 2% milk Day 4 1 CheeseCroissant 250 ml. Sliced Peaches in syrup 300 ml. Orange juice 2Flatbread Sammie, with Chicken, bacon, cheddar, tomato & romaine lettuceOn side Buttermilk ranch dressing Cup Broccoli & Cheese soup 1-pkg.Crackers 355 ml. Ginger ale 425 g. Beef Stew, tender Beef cubes, withcar- rots, onions, & potatoes 325 g. Thai salad, with romaine lettuce,pasta, & dressings 1 Italian bread 250 ml. Sliced Pears in syrup 300 ml.Apple juice 1 Blueberry muffin 250 ml. 2% Milk Day 5 375 ml. Corn FlakesMed. Banana 250 ml. 2% Milk 300 ml. Apple juice 6 pcs. Chicken wings 250ml. Fried rice 250 ml. Mixed cooked veggies, green beans, car- rots, &red peppers with sundried tomato sauce 355 ml. 7-up 6″ Pizza Pepperonilovers Pizza a double helping of deli style pepperoni & 100% PizzaMozzarella Small Salad 355 ml. Ginger Ale 1 Almond Croissant 250 ml. 2%Milk Day 6 45 g. Bagel with Cream Cheese [Discharge] 300 ml. Orangejuice Day 22 450 g. Breakfast Omelets Spinach & Feta cheese 2 slicesBrown toast 300 ml. Orange juice  9″ Zesty Grille Steak Prime Rib Steak,mozzarella, cheddar, mushroom, saut{acute over ()}éed onion, on Cheesebread On side Honey Bourbon Mustard, Zesty Grille Sauce Cup Broccoli &Cheese soup 1-pkg. Crackers 355 ml. Ginger Ale 400 g. Herb GrilledChicken Breast with Vegetables 1.5 cups Flavored rice Small Fresh fruitsalad 300 ml. Apple juice 1 Raisin Oatmeal cookie 250 ml. 2% Milk Day 2345 g. Butter croissant [Discharge] 300 ml. Orange juice

Results

Overall, ISIS 304801 demonstrated a good safety profile and was welltolerated in all subjects with no clinically meaningful elevations oftransaminase enzymes and no significant adverse events.

The baseline characteristics of MAD cohorts are shown in Table 34. MADsubjects showed dose-dependent sustained reductions in total apoC-IIIand TG levels expressed as a percentage change from baseline in Tables35-36.

TABLE 34 Baseline Characteristics of MAD Cohorts Placebo 50 mg 100 mg200 mg 400 mg (n = 4) (n = 3) (n = 3) (n = 3) (n = 3) Gender (M:F) 3:13:0 3:0 3:0 3:0 Age (yrs) 43.0 40.0 40.0 43.0 40.0 BMI (kg/m2) 27.7 24.027.3 28.0 27.5 Lipids & Lipoproteins, mg/dL Apo CIII 6.3 10.4 9.5 11.68.7 Triglycerides 97 124 94 195 89 Total Cholesterol 195 157 196 185 181HDL-C 45 42 46 43 62 Non-HDL-C 136 118 150 149 126 LDL-C 112 93 131 95102 Per-protocol population. Values presented are the median.

TABLE 35 Dose-Dependent Prolonged Reduction in Serum ApoCIII: Median %change in ApoCIII from Baseline Study Day Placebo 50 mg 100 mg 200 mg400 mg Day 5 38.6 33.4 −4.5 −10.8 −42.6 Day 8 31.9 17.8 −5.2 −36.0 −78.6Day 15 0.0 −22.1 −24.0 −54.1 −79.8 Day 22 11.7 −20.9 −21.0 −61.7 −86.4Day 23 15.8 −15.6 −11.9 −58.7 −79.0 Day 29 −11.0 −19.7 −17.3 −70.5 −77.5Day 36 1.9 −26.6 −32.1 −57.3 −69.5 Day 50 16.4 21.9 −3.9 −63.0 −78.6

TABLE 36 Dose-Dependent Reductions in Triglycerides: Median % Change inTriglycerides from Baseline Study Day Placebo 50 mg 100 mg 200 mg 400 mgDay 5 78.5 50.0 15.9 −15.6 −7.7 Day 8 34.9 17.7 −24.5 −31.8 −38.5 Day 1521.2 −27.4 −12.8 −50.8 −46.2 Day 22 12.2 −18.3 −9.8 −25.1 −53.8 Day 2351.8 −4.0 1.1 −41.0 −44.2 Day 29 28.5 −19.5 −25.0 −43.1 −43.8 Day 3615.4 −33.1 −34.8 −17.9 −36.5 Day 50 33.1 48.8 −23.9 −48.2 −48.1

Median percent change from baseline values in the 50, 100, 200 and 400mg multiple-dose groups showed reductions of total apoC-III of 20, 17,71, and 78% and of TG of 20, 25, 43, and 44%, respectively, one week(Day 29) after the last dose. Reductions were sustained for at leastfour weeks after the last dose in the higher dose groups.

TG levels spiked at Day 5 and 23 for the placebo group, coinciding withthe subjects' overnight stays at the Study Center. It is thought thatthe diet provided by the Study Center led to the surge in TG levels inthe subjects staying overnight at the Study Center. ISIS 304801decreased the TG spike in a dose-dependent manner. In a manner, theresults shown herein, indicate a postprandial effect on TG (although TGlevels were assessed after a 12 hour fast) by ISIS 304801 as a dietinduced surge in TG was decreased in a dose-dependent manner by ISIS304801.

LDL-C values did not change (data not shown) while HDL-C values tendedto increase in a treatment-dependent manner as shown in Table 37.

TABLE 37 No Deleterious Effects on HDL-C Study Day Placebo 50 mg 100 mg200 mg 400 mg Day 5 −5.9 7.7 −3.1 −3.2 −16.1 Day 8 2.1 2.4 0.0 −11.1−2.9 Day 15 −2.8 4.8 10.9 4.7 −9.7 Day 22 −0.6 4.8 8.7 11.6 −2.0 Day 230.7 7.1 12.5 19.4 −1.6 Day 29 2.1 19.0 0.0 13.9 8.0 Day 36 0.0 23.8 8.813.9 1.6 Day 50 −4.8 16.7 5.9 25.0 14.5

Example 11 ISIS 304801 Phase II Clinical Trial

A randomized, double-blind, placebo-controlled, dose response study isplanned to evaluate the dose/response pharmacodynamic effects of ISIS304801 vs. placebo on fasting apoC-III associated with VLDL levels.Additional endpoints to evaluate include: the pharmacodynamic effects ofISIS 304801 vs. placebo on fasting total apoC-III, TG, apoC-II (totaland associated with VLDL), apolipoprotein B-100 (apoB-100),apolipoprotein A-1 (apoA-1), apolipoprotein A-2 (apoA-2), apolipoproteinE (apoE), total cholesterol (TC), low-density lipoprotein-cholesterol(LDL-C), LDL-TG, VLDL-C, VLDL-TG, non-high-densitylipoprotein-cholesterol (non-HDL-C), non-HDL-TG, HDL-C, HDL-TG,chylomicron-C(CM-C), CM-TG, free fatty acids (FFA), and glycerol levels;the post-prandial lipid, apolipoprotein and lipoprotein characteristicsand kinetics, and glucose levels in a subset of the patients in thestudy and assess more extensive PK in another subset of the patients(will not be the same patients as those undergoing the post-prandialassessment); and, the safety, tolerability and PK of ISIS 304801.

For each patient, the participation period consists of a ≦5-weekscreening period, (which includes a 4-week tight diet control run-inqualification period), a 1-week study qualification/baseline assessmentperiod, a 13-week treatment period, and a post-treatment evaluationperiod of 13 weeks, for a total of 32 weeks of study participation.Concomitant medications and adverse events (AEs) will be recordedthroughout all periods of the study.

Patients will be at least 18 years of age and have fasting TG≧500 mg/dLat screening and fasting TG≧300 mg/dL and ≦2000 mg/dL after 4-weeks oftight diet control run-in.

Seventy two (72) patients are planned for this study. There will be 24patients planned per dose cohort (100, 200, 300 mg) with 18 ISIS 304801(active) and 6 placebo patients per cohort. Eligible patients will beenrolled equally (1:1) into a non-extensive PK/post-prandial group(Group 1) or an extensive PK/post-prandial group (Group 2). Patients inGroup 2 will be randomized equally (1:1) to an extensive PK group (Group2a) or a post-prandial assessment group (Group 2b). Group 2a patientswill be randomized equally (1:1:1) to 1 of the 3 dose cohorts (100, 200,300 mg) and, within each dose cohort, 5:1 to receive active or placebo.Group 2b patients will be randomized equally (1:1) to 1 of 2 dosecohorts (200, 300 mg) and, within each dose cohort, 2:1 to receiveactive or placebo. Group 1 patients will be randomized to dose cohortand treatment in a manner that achieves an overall study randomizationof 1:1:1 to dose cohort (100, 200, 300 mg) and 3:1 to treatment (active,placebo).

Patients will be placed on a tightly controlled diet (after screeningprocedures are performed) for the duration of study participation. After28 days on the controlled diet, patients will have baseline measurementsand be assessed for qualification of enrollment into the treatment phaseof the study. Patients who meet the enrollment criteria following dietrun-in will be enrolled equally (1:1) into a non-extensivePK/post-prandial group (Group 1) or into an extensive PK/post-prandialgroup (Group 2) and randomized within their Group assignment.

Study Drug and Treatment

A solution of ISIS 304801 (200 mg/mL, 1.0 mL) contained in 2-mLstoppered glass vials will be provided.

The placebo for this study will be 0.9% sterile saline.

ISIS 304801 solution and placebo will be prepared by an unblindedpharmacist (or qualified delegate). Vials are for single-use only. Atrained professional, blinded to the identity of the drug, willadminister the Study Drug. The Study Drug will be administered as a SCinjection in the abdomen, thigh, or outer area of the upper arm on eachdosing day. Doses of 100 and 200 mg will be administered as a single SCinjection. Doses of 300 mg will be administered as two equal volumenoncontiguous SC injections.

Patients will receive 13 doses of Study Drug administered by SCinjection once a week for 13 weeks (Days 1, 8, 15, 22, 29, 36, 43, 50,57, 64, 71, 78, and 85).

Patients will complete the treatment visits on Day 1±0 days and on Day8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85 within ±1 day.Patients in the extensive PK group will also visit the clinic on Day 2and 86±0 days relative to Day 1 and 85, respectively, for the 24 hourblood draw. Patients will complete the follow-up visits on Day 92 and 99within ±1 day, Day 127 within ±3 days, and Day 176 within ±5 days of thescheduled visit date. Patients in the post-prandial assessment groupwill also visit the clinic on Day 103 within ±2 days and on the dayfollowing the Day 103 visit for the 24 hour blood draw.

Preceding each visit that includes a blood draw for pharmacodynamicmeasurements (Days 8, 15, 29, 43, 57, 71, and 85), patients will beprovided a standardized pre-cooked meal for the dinner on the eveningprior to their visit (to ensure equal moderation of fat intake, perpatient and per time point) after which they will remain fasted. Alcoholconsumption will not be allowed for 48 hrs preceding these clinicvisits.

Blood will be collected for measurement of VLDL apoC-III and otherpharmacodynamic markers on Days 8, 15, 29, 43, 57, 71, and 85 (prior toStudy Drug administration).

Patients in the post-prandial assessment group will consume standardizedpre cooked meals (lunches and dinners (provided) and instructions forbreakfasts and snacks) for the 2 days prior to the post-prandialevaluations. On each of the post-prandial evaluation days, following theblood draws, patients will consume a standardized liquid meal, whichrepresents about a third of the daily caloric requirements, with astable radioisotope tracer, followed by serial blood sampling. Patientswill receive a standardized pre-cooked meal 9 hrs after consuming theliquid meal, after which they will fast until the 24 hour blood draw thefollowing day.

In addition to trough sample collection, patients in the extensive PKassessment group will undergo serial blood sampling for 24 hrs aftertheir first (Day 1-2) and last (Day 85-86) dose of Study Drug.

Post-Treatment Evaluation Period

Patients will be followed until Study Day 176. During this time,patients will return to the study center for outpatient clinic visits onStudy Days 92, 99, 127, and 176 (and Day 103 for patients in thepost-prandial assessment group) for safety and clinical laboratoryevaluations (blood draws), diet counseling and monitoring, concomitantmedication usage recording, and AE event collection.

Blood samples for PK and PD analysis will be collected periodicallythroughout the post-treatment evaluation period. Laboratory measurementsof serum chemistry, urinalysis, coagulation, complement, hematology,immune function, thyroid function, and full lipid panel will beperformed at the various times throughout the study.

Post-prandial assessments will be done in a subset of patients asdescribed below.

Post-Prandial Meal, Sampling Schedule, and Assessment

Post-prandial assessment for lipoproteins metabolism will be performedusing a radiolabelled meal supplemented with a labeled tracer,3H-palmitate (300 μCi, Perkin Elmer Inc., Woodbridge, ON, Canada),sonicated into the liquid meal. Palmitate is a fatty acid that is acommon constituent of any diet. The 3H-palmitate tracer emits weakradioactivity, equivalent to an X-ray. Since dietary palmitate isincorporated into chylomicrons as they are formed in the enterocytes ofthe gut, this enables monitoring the appearance and clearance ofnewly-formed chylomicrons from circulation. The methodology to beapplied for studying post-prandial kinetics of chylomicrons appearanceand clearance is well-established (Mittendorfer et al. 2003, Diabetes,52: 1641-1648; Bickerton et al. 2007; Normand-Lauziere et al. 2010,PLoS. One, 5: e10956).

A liquid meal (similar to a milkshake) containing a small amount (300μCi) of radiolabelled fatty acids (3H-palmitate) will be provided. Theliquid meal will provide about a third of the daily caloricrequirements. From 1 hr prior to 9 hrs after the ingestion of the meal,a constant infusion of [U-13C]—K palmitate (0.01 μmol/kg/min in 100 ml25% human serum albumin; Cambridge Isotopes Laboratories Inc., Andover,Mass.) and a primed (1.6 μmol/kg) continuous (0.05 μmol/kg/min) infusionof [1,1,2,3,3-2H]-glycerol (Cambridge Isotopes Laboratories Inc.) willbe administered as previously described (Normand-Lauziere et al. 2010,PLoS. One, 5: e10956). Plasma palmitate and glycerol appearance rateswill be calculated using Steele's non-steady state equation assuming avolume of distribution of 90 ml/kg and 230 ml/kg, respectively(Gastaldelli et al. 1999, J Appl. Physiol, 87: 1813-1822).

Blood samples will be drawn at intervals before and after the ingestionof the radiolabelled meal on days prior to and after the Treatment phaseas noted in the table below. A standardized meal will be given to theparticipants after the 9 hr blood draw. Blood will be collected in tubescontaining Na2 EDTA and Orlistat (30 μg/ml, Roche, Mississauga, Canada)to prevent in vitro triacylglycerol lipolysis and separate samples willbe collected in NaF tubes for plasma glucose determination.

The following will be measured at each time-point:

-   -   Plasma and CM fraction levels for 3H-tracer    -   Plasma [U-13C]—K palmitate and [1, 1, 2, 3, 3-2H]-glycerol        appearance rates    -   Plasma and CM fraction levels for TG, TC, and apoB    -   Plasma and VLDL fraction levels for apo CIII, apo CII, and apo E    -   Plasma levels for glucose

Plasma samples may also be used for profiling of drug binding proteins,bioanalytical method validation purposes, stability and metaboliteassessments, or to assess other actions of ISIS 304801 with plasmaconstituents.

1. A method of increasing high density lipoprotein (HDL) levels in ahuman subject, comprising (a) selecting a human subject in need oftreatment of cardiovascular disease, and (b) administering to the humansubject a therapeutically effective amount of a compound comprising amodified oligonucleotide 12 to 30 nucleobases in length targeted to anapolipoprotein C-III (ApoCIII) nucleic acid as shown in SEQ ID NOs:1-2,wherein after (b) HDL levels are increased and the cardiovasculardisease is treated in the human subject. 2.-14. (canceled)
 15. Themethod of claim 1, wherein the cardiovascular disease ishypertriglyceridemia or dyslipidemia. 16.-23. (canceled)
 24. The methodof claim 1, wherein the modified oligonucleotide has a nucleobasesequence comprising at least 8 contiguous nucleobases of a nucleobasesequence of SEQ ID NO:
 3. 25. The method of claim 1, wherein thenucleobase sequence of the modified oligonucleotide is at least 80%, atleast 90% or 100% complementary to a nucleobase sequence of SEQ ID NO: 1or SEQ ID NO:
 2. 26.-27. (canceled)
 28. The method of claim 1, whereinthe modified oligonucleotide consists of a single-stranded modifiedoligonucleotide.
 29. (canceled)
 30. The method of claim 1, wherein themodified oligonucleotide consists of 20 linked nucleosides.
 31. Themethod of claim 1, wherein at least one internucleoside linkage of themodified oligonucleotide is a modified internucleoside linkage.
 32. Themethod of claim 31, wherein at least one modified internucleosidelinkage of the modified oligonucleotide is a phosphorothioateinternucleoside linkage.
 33. The method of claim 1, wherein at least onenucleoside of the modified oligonucleotide comprises a modified sugar.34. The method of claim 33, wherein at least one modified sugar is abicyclic sugar.
 35. The method of claim 33, wherein at least onemodified sugar comprises a 2′-O-methoxyethyl.
 36. The method of claim 1,wherein at least one nucleoside of the modified oligonucleotidecomprises a modified nucleobase.
 37. The method of claim 36, wherein themodified nucleobase is a 5-methylcytosine. 38.-39. (canceled)
 40. Themethod of claim 30, wherein the modified oligonucleotide comprises: (a)a gap segment consisting of 10 linked deoxynucleosides; (b) a 5′ wingsegment consisting of 5 linked nucleosides; (c) a 3′ wing segmentconsisting 5 linked nucleosides; wherein the gap segment is positionedimmediately adjacent to and between the 5′ wing segment and the 3′ wingsegment and wherein each nucleoside of each wing segment comprises a2′O-methoxyethyl sugar, wherein each cytosine is a 5′-methylcytosine,and wherein each internucleoside linkage is a phosphorothioate linkage.41. The method of claim 40, wherein the modified oligonucleotide has anucleobase sequence comprising at least 8 contiguous nucleobases of anucleobase sequence of SEQ ID NO:
 3. 42.-85. (canceled)
 86. The methodof claim 1, wherein the HDL levels are increased by at least 20% in thehuman subject.
 87. The method of claim 1, wherein the nucleobases of themodified oligonucleotide consist of the nucleobase sequence of SEQ IDNO:
 3. 88. The method of claim 1, wherein the compound comprises aconjugate.
 89. The method of claim 1, wherein the compound is in a saltform.
 90. A method for treating chylomicronemia in a human subject,comprising (i) selecting a human subject in need of treatment ofchylomicronemia, and (ii) administering to the human subject atherapeutically effective amount of a compound comprising a modifiedoligonucleotide 12 to 30 nucleobases in length targeted to anapolipoprotein C-III (ApoCIII) nucleic acid as shown in SEQ ID NO: 1-2,wherein administration of the compound to the human subject treats thechylomicronemia.
 91. The method of claim 90, wherein the modifiedoligonucleotide consists of a single-stranded modified oligonucleotide.92. The method of claim 90, wherein the modified oligonucleotide has anucleobase sequence comprising at least 8 nucleobases of the nucleobasesequence of SEQ ID NO:
 3. 93. The method of claim 90, wherein themodified oligonucleotide consists of 20 linked nucleosides.
 94. Themethod of claim 93, wherein the nucleobases of the modifiedoligonucleotide consist of the nucleobase sequence of SEQ ID NO:
 3. 95.The method of claim 94, wherein the modified oligonucleotide comprises:(a) a gap segment consisting of 10 linked deoxynucleosides; (b) a 5′wing segment consisting of 5 linked nucleosides; (c) a 3′ wing segmentconsisting 5 linked nucleosides; wherein the gap segment is positionedimmediately adjacent to and between the 5′ wing segment and the 3′ wingsegment, wherein each nucleoside of each wing segment comprises a2′-O-methoxyethyl sugar, wherein each cytosine is a 5′-methylcytosine,and wherein each internucleoside linkage is a phosphorothioate linkage.96. The method of claim 90, wherein the compound comprises a conjugate.97. The method of claim 90, wherein the compound is in a salt form. 98.A method for treating pancreatitis in a human subject, comprising (i)selecting a human subject in need of treatment of pancreatitis, and (ii)administering to the human subject a therapeutically effective amount ofa compound comprising a modified oligonucleotide 12 to 30 nucleobases inlength targeted to an apolipoprotein C-III (ApoCIII) nucleic acid asshown in SEQ ID NO: 1-2, wherein administration of the compound to thehuman subject treats the pancreatitis.
 99. The method of claim 98,wherein the modified oligonucleotide consists of a single-strandedmodified oligonucleotide.
 100. The method of claim 98, wherein themodified oligonucleotide has a nucleobase sequence comprising at least 8nucleobases of the nucleobase sequence of SEQ ID NO:
 3. 101. The methodof claim 98, wherein the modified oligonucleotide consists of 20 linkednucleosides.
 102. The method of claim 101, wherein the nucleobases ofthe modified oligonucleotide consist of the nucleobase sequence of SEQID NO:
 3. 103. The method of claim 102, wherein the modifiedoligonucleotide comprises: (a) a gap segment consisting of 10 linkeddeoxynucleosides; (b) a 5′ wing segment consisting of 5 linkednucleosides; (c) a 3′ wing segment consisting 5 linked nucleosides;wherein the gap segment is positioned immediately adjacent to andbetween the 5′ wing segment and the 3′ wing segment, wherein eachnucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar,wherein each cytosine is a 5′-methylcytosine, and wherein eachinternucleoside linkage is a phosphorothioate linkage.
 104. The methodof claim 98, wherein the compound comprises a conjugate.
 105. The methodof claim 98, wherein the compound is in a salt form.